Research in Veterinary Science 93 (2012) 675–681

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Research in Veterinary Science

journal homepage: www.elsevier .com/locate / rvsc

Oral immunisation of mice with a recombinant rabies virus vaccineincorporating the heat-labile enterotoxin B subunit of Escherichiacoli in an attenuated Salmonella strain

Xuelin Wang a,1, Juan Liu a,1, Xiuping Wu a,1, Lu Yu a,1, Haiying Chen a,1, Heng Guo a,1, Maolin Zhang a,Huiping Li a, Xue Liu a, Shumin Sun a,b, Lijing Zhao a, Xinyue Zhang a, Lifang Gao a,⇑, Mingyuan Liu a,⇑a Key Laboratory of Zoonosis, Ministry of Education, Institute for Zoonosis, Jilin University, 5333 Xian Road, 130062 Changchun, PR Chinab Animal Science and Technology College, Inner Mongolia University for Nationalities, 028000 Tongliao, PR China

a r t i c l e i n f o

Article history:Received 17 May 2011Accepted 19 September 2011

Keywords:RabiesVaccinesImmunitySalmonellaCVS-11

0034-5288/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.rvsc.2011.09.015

⇑ Corresponding authors. Tel./fax: +86 431 8783670E-mail address: liumy@jlu.edu.cn (M. Liu).

1 These authors equally contribution to this paper.

a b s t r a c t

To investigate effective new rabies vaccines, a fusion protein consisting of the rabies virus (RV) glycopro-tein and the heat-labile enterotoxin B subunit of Escherichia coli (LTB) was successfully constructed anddelivered in a live attenuated Salmonella strain LH430. Mice were immunised with LH430 carryingpVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The antibody titres of mice immunised with oral LH430carrying pVAX1-G-LTB or pVAX1-ori-G-LTB were significantly higher than those of pVAX1-G-immunisedmice. The results of the challenge with the rabies virus standard strain (CVS-11) showed that the LH430strain carrying the G-LTB gene induced immunity and elevated IL-2 levels in immunised mice (⁄⁄P < 0.01),whereas LH430 carrying pVAX1-G did not contribute to protection. These results show that LH430 car-rying recombinant G-LTB could provide overall immunity against challenge with CVS-11 and shouldbe considered to be a potential rabies vaccine.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction Enhancing the immunogenicity of DNA-based rabies vaccines is

Rabies is an important viral disease (Jackson, 2008), and morethan 60,000 people die from rabies each year in Africa and Asia(WHO, 2007; Dodet et al., 2008; Osinubi et al., 2009). In China,rabies killed 3293 people in 2006, which was 27% more than in2005 (WHO, 2007). It has been estimated that the cost of rabiesvirus (RV) vaccination is almost $600 million per year and is risingin Africa and Asia (Knobel et al., 2005).

In China, cell-culture-derived inactivated rabies vaccines andthe live attenuated Evelyn Rokitnicki Abelseth (ERA) vaccine havebeen used to vaccinate dogs. However, the expense of these vac-cines limits their use in developing countries. Furthermore, liveattenuated vaccines may revert to virulence in immunised dogs(Fehlner-Gardiner et al., 2008; Müller et al., 2009). The low immu-nogenicity of DNA-based rabies vaccines reduces their utility asclinical application and undermines the ability of this approachto induce adequate quantities of viral-neutralising antibodies(VNAs). Consequently, rabies remains prevalent in dogs and catsin developing countries (Ogunkoya et al., 1990; Bahloul et al.,2006), and, therefore, a safe and immunogenic rabies vaccinewould be of benefit in Asia and Africa (Gupta et al., 2009).

ll rights reserved.

2.

an emerging strategy and has profound effects on the efficacy ofvaccines in preclinical models. The heat-labile enterotoxin B sub-unit of Escherichia coli (LTB) has been shown to have immunomod-ulatory activity. Thus, LTB has been considered as a mucosal andparenteral adjuvant for enhancing the immunogenicity of vaccinesin different animal models (Simmons et al., 2001).

Heterologous antigens fused with the LTB gene in attenuatedSalmonella spp. have been used to induce humoral and cellular im-mune responses (Qiao et al., 2009; da Hora et al., 2011; Hur et al.,2011). The oral administration of LTB has been shown to promoteheterologous protein exposure to the immune system by bindingwith galactosyl-N-acetylgala-ctosamylsialyl-galactosylglucosylcera-mide (GM1) ganglioside anchored on the surface of intestinal epi-thelial cells, thereby producing high titres of serum antibodies inmice (Liu et al., 2011). LTB activates B and CD4+ T cells through inter-actions between the B subunit pentamers of LTB and the GM1 recep-tor on B and CD4+ T cells and also strengthens the presentation ofantigens by activating dendritic cells (DCs) and other antigen-presenting cells by facilitating antigen uptake through a receptor-mediated endocytosis mechanism (Nashar et al., 2001). TheGM1-mediated presentation of LTB by B cells and CD11c + DCssignificantly enhances the proliferation and cytokine expression ofLTB-specific CD4+ T cells. LTB presentation by B cells also provideshelper signals for the induction of antigen-specific immunoglobulinB cells. Furthermore, GM1-mediated interactions can significantly

676 X. Wang et al. / Research in Veterinary Science 93 (2012) 675–681

enhance the presentation of LTB-conjugated antigens (Ertl, 2009).LTB was used in this study because it can carry foreign proteins.

2. Materials and methods

The animal experiments were performed in compliance withthe guidelines of the local ethics committee, and the study was ap-proved by the Institutional Review Board at Jilin University.

This work was undertaken from August to November 2010.

2.1. Cells and mice

Baby hamster kidney (BHK)-21 cells stored by the Institute forZoonosis of Jilin University were maintained in Minimum EssentialMedium (MEM) (Gibco, USA) containing 5% heat-inactivated foetalbovine serum (FBS) (Gibco, USA), 100 U/ml penicillin (GE, USA) and100 g/ml streptomycin (GE, USA) in 37 �C, 5% CO2. Six- to eight-week-old female BALB/c mice (provided by Changchun H&N Ani-mal Breeding Centre for Medicine, Changchun, China) were treatedaccording to institutional guidelines.

2.2. Virus, bacterial strains and plasmids

The rabies virus standard strain CVS-11, which was purchasedfrom the Centre for Disease Control of China, was recovered byinoculation of mouse brains. CVS-11 was used to determine themedian lethal dose (LD50) in sucking BALB/c mice (14–16 g). Thehighly attenuated Salmonella typhimurium (ST) LH430 strains(phoP, phoQ) were generously donated by the College of BasicMedicine, Jilin University. The pVAX1-G and pVAX1-ori plasmidswere constructed and maintained in our laboratory.

2.3. Recombinant plasmid construction

The rabies virus CTN-30 glycoprotein gene (GenBank accessionnumber DQ767895) and LTB gene (GenBank accession numberFJ156281) were used to design the primers and amplify the glyco-protein (G) and LTB genes. The fusion gene G-LTB was constructedvia gene splicing by overlap extension (SOE) PCR using the linker50-GCTGCCACCGCCACCGCTGCCACCGCCACC-30. The procedurewas as follows: DNA fragments encoding the G and LTB proteinswithout the signal peptide sequence were isolated by PCR usingthe primers P1: 50-CGGGATCCAAATTCCCTATTTACACGATACC-30,P2: 50-CCGCTGCCACCGCCACCGCTGCCACCGCCACCTCTAGACAGCTTGGTCTCACCTCCG-30, P3: 50-AGCGGTGGCGGTGGCAGCGGTGGCGGTGGCAGCCTGCAGGCTCCCCA GACTATTACAG-30, and P4: 50-CCCTCGAGCTAGTTTTTCATACTGATTGCC-30. The products were purifiedusing Axygen’s PCR products purification system (Axygen, China)and were used as the template for the synthesis of G-LTB. Theprimers P1: 50-CGGGATCCAAATTCCCTATTTACACGATACC-30 andP4: 50-CCCTCGAGCTAGTTTTTCATACTGATTGCC-30 were used inthe second PCR. Purification was performed as described above.The product was cloned into the pVAX1 (Invitrogen, USA) andpVAX1-ori plasmids using the restriction enzymes BamHI andXhoI. The plasmids pVAX1, pVAX1-G, pVAX1-G-LTB, and pVAX1-ori-G-LTB with ori replication origin were electroporated intoLH430 using a cuvette (Bio-Rad, USA).

2.4. Antigenicity analysis of the recombinants

To analyse the expression of RV-G, the plasmids pVAX1, pVAX1-G, pVAX1-G-LTB, and pVAX1-ori-G-LTB were transfected intoBHK-21 cells. The transfected cells were harvested, fixed with 4%paraformaldehyde for 20 min at room temperature and treatedwith 0.1% Triton X-100. After washing with PBS three times, the

cells were co-incubated with a mouse anti-rabies glycoproteinmonoclonal antibody (Santa Cruz, USA) (1:400) at 37 �C for 1 h.Next, the cells were incubated with fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse IgG (Santa Cruz, USA) (1:500).Indirect immunofluorescence and green fluorescence signals wereobserved by confocal laser scanning microscopy (Olympus, Japan).

2.5. Western blot analysis of the recombinants

BHK-21 cells (1 � 107) were seeded in tissue culture flasks andwere transfected with pVAX1, pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB at 37 �C for 48 h. After transfection, the cells were col-lected and lysed with lysis buffer (10 mM Tris–HCl, pH 7.4,150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) to collectthe protein. The protein was eluted with 50 ll of 0.1 M diethanola-mine and was resolved by sodium dodecyl sulphate–12% polyacryl-amide gel electrophoresis. The isolated proteins were transferredonto a nitrocellulose membrane, and a mouse anti-RV monoclonalantibody (Santa Cruz, USA) (1:400) and an alkaline phosphatase(AP)-conjugated goat anti-mouse IgG (H + L) (Proteintech Group,USA) (1:1000) were used as the primary and secondary antibodies,respectively, according to the manufacturers’ instructions. Afterthe membranes had been treated with enhanced chemilumines-cence solution (Pierce, USA), the protein bands were visualised byX-ray film exposure (Kodak, China) (Shah et al., 2011).

2.6. Immunisation of mice

ST were grown in lysogeny broth (LB) medium with appropriatesupplements at 37 �C. The bacteria were harvested, and 5 �1010 CFU/ml of the ST were used to immunise mice. All experimen-tal mice were deprived of food and water for 4 h prior to immuni-sation. Five randomised subgroups consisting of 10 mice per groupwere orally immunised with 200 ll LH430 harbouring pVAX1-G,pVAX1-G-LTB, pVAX1-ori-G-LTB or pVAX1 on days 0 and 10, andthe control group was given PBS. After immunisation, sera werecollected on days 0, 10, 21, 35 and 45 for further testing.

2.7. Plasmid stability in vitro

Transformed Salmonella strains were cultured in LB medium at37 �C overnight, passaged using a 1:100 dilution in fresh mediumwith and without antibiotics and observed every 6 h for 24 h.CFU were determined by serial dilution. The stability was calcu-lated based on the ratio of the CFU grown on antibiotic agar platescontaining 50 lg/ml kanamycin to the total CFU grown on LB-agarwithout selecting antibiotics.

2.8. Plasmid stability in vivo

After mice were orally immunised with the different recombi-nant-protein-expressing LH430 strains and batched, their livers,spleens and Peyer’s patches (PPs) were removed at 1, 3 or 7 d andhomogenised in PBS containing 0.1% Triton X-100. The homogenisedtissue samples were handled using a Polytron PT 3000 homogeniser(Kinematica AG, Littau-Lucerne, Switzerland) to release the intracel-lular bacteria. Serial dilutions were streaked on LB-agar plates withor without 50 lg/ml kanamycin to determine the extent of organcolonisation and to calculate the plasmid stability.

2.9. IgG induction by the recombinant LH430

The immunoglobulin levels were determined using a mouseanti-RV antibody ELISA kit (Boster, China). After microtitrationplates were coated with mouse anti-RV monoclonal antibody andincubated at 4 �C overnight, they were co-incubated with a 1:10

Fig. 1. Recombinant construction. (A) Amplification fragments of the LTB, G and G-LTB fusion genes by PCR. Lane M: marker; lane 1: amplification fragment of the LTB gene;lane 2: amplification fragment of the G-LTB fusion gene; and lane 3: amplification fragment of the G gene. (B) Digestion analysis of the pVAX1-G-LTB plasmid using doubledigests. Lane M: marker; lane 1: pVAX1-G-LTB digested with PstI and XhoI; lane 2: pVAX1-G-LTB digested with BamHI and XhoI; and lane 3: pVAX1-G-LTB. (C) Digestionanalysis of pVAX1-ori-G-LTB using double digests. Lane M: marker; lane 1: pVAX1-ori-G-LTB digested with BamHI, XhoI; lane 2: pVAX1-ori-G-LTB digested with BamHI, XbaI;and lane 3: pVAX1-ori-G-LTB.

Fig. 2. Fluorescence microscopy analysis of the expression of recombinant G-LTB. (A): Non-transfected BHK-21 cells; (B): pVAX1-transfected BHK-21 cells; (C): pVAX1-G-transfected BHK-21 cells; (D): pVAX1-G-LTB-transfected BHK-21 cells; and (E): pVAX1-ori-G-LTB-transfected BHK-21 cells. Green fluorescence represents successful G-LTBexpression in BHK-21 cells. Bright green signals were observed in panels C, D and E (20 � 10).

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dilution of either pre- or post-vaccination mouse serum samples at37 �C for 30 min. The plates were washed with PBS containing0.05% Tween-20 (Ding guo, China) five times and were subse-quently incubated with horseradish peroxidase (HRP)-conjugatedgoat anti-mouse IgG (R&D, USA) at 37 �C for 30 min. Followingthe reactions, all plates were washed as described previously andwere incubated with substrate solution. Finally, the reaction wasstopped with 100 ll of stop solution. The absorbance was read at450 nm. The antibody levels of the vaccinated mice were repre-sented as the geometric mean of the absorbance.

2.10. Detection of neutralising antibodies in immunised mice

To perform the FAVN test, BHK-21 cells and the RABV strain CVS-11 were used. The positive control serum against RABV was obtainedfrom adult dogs vaccinated with a rabies vaccine. Next, the serum

was diluted to 0.5 IU/ml and used as a standard serum. Serialthree-fold dilutions of the positive and negative control sera andsera from mice immunised with 200 ll LH430 harbouring pVAX1-G, pVAX1-G-LTB, pVAX1-ori-G-LTB or pVAX1 on days 0 and 10 weremade in 100 ll volumes in microplate wells. Each sample was addedto four adjacent wells and serially diluted five times using a multi-channel pipette. Fifty-microlitre challenge virus suspensions con-taining 100 TCID50 were added to each well. The microplates wereincubated for 1 h at 37 �C in a humidified incubator containing 5%CO2. Following incubation, 2 � 104 BHK-21 cells in 50 ll of culturemedium were added to each well, and the plates were incubatedfor another 48 h. Next, the medium was discarded, and the plateswere rinsed in PBS (pH 7.2) and acetone (80% in distiled water).The plates were subsequently fixed in 80% acetone for 30 min atroom temperature and air-dried. The staining was carried out byadding 50 ll of fluorescein isothiocyanate-conjugated anti-rabies

Fig. 3. Western blot analysis of BHK-21 cells transfected with pVAX1, pVAX1-G,pVAX1-G-LTB, or pVAX1-ori-G-LTB. Lane M: marker; lanes 1–4: BHK-21 cellstransfected with pVAX1, pVAX1-G, pVAX1-G-LTB, and pVAX1-ori-G-LTB, respec-tively; and lane 5: non-transfected BHK-21 cells.

Fig. 4. The stability of the plasmid in the recombinant LH430 strain during culture.The plasmid stability in the bacterium was assayed. Values represent the average ofthree similar plasmids per data point (mean ± S.D.). The experiment was performedtwice with similar results.

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serum. After incubation for 30 min at 37 �C, the plates were washedtwice with PBS. The FAVN result was assessed with a fluorescencemicroscope (Zeiss, Germany). The total area of each well was exam-ined. The well was considered positive if one or more fluorescentcells were observed; otherwise, it was considered negative. The50% endpoint of the antibody content of the test sample and virus ti-tres (TCID50) were calculated according to the Spearman-Karbermethod (Cliquet et al., 1998). The neutralising titre was calculatedbased on the number of negative wells and their dilution factors rel-ative to those of the standard serum included in the assay.

2.11. Challenge of immunised mice with CVS-11

On day 10, all mice were challenged intracerebrally with 30 llof 20 LD50 of CVS-11. All challenged mice were observed for theappearance of rabies-specific signs or death for 14 d, and the re-sults were confirmed with a final acceptance test (FAT). Finally,the survival rate was calculated.

2.12. Serum IL-2 levels of the immunised mice

The IL-2 levels were determined using a mouse IL-2 cytokineELISA kit (Boster, China). According to the manufacturer’s proto-col, a 96-well microtiter ELISA plate was coated with anti-IL-2cytokine antibody and was incubated with sera of the immunised

mice at 37 �C for 1.5 h. The plate was dried, washed five timesand incubated with HRP-conjugated anti-mouse IL-2 antibody(R&D, USA) at 37 �C for 1 h. Next, the plate was washed as beforeand reacted with 100 ll of substrate solution at 37 �C in the darkfor 15 min. The reaction was stopped by adding 100 ll of stopsolution to each well. The absorbance was read at 450 nm usinga microplate reader (Bio-Rad, USA) within 30 min. The concentra-tions of IL-2 in all sera were calculated using the absorbance val-ues of the standard sample provided by the manufacturer (R&D,USA).

2.13. Statistical analysis

All data were analysed with the SPSS 13.0 Data Editor (SPSS Inc.,Chicago, Illinois, USA). Statistical significance was determined bythe v2 test, with P < 0.05 considered statistically significant.

3. Results

3.1. Recombinant protein construction

Three recombinant plasmids were successfully constructed, andthe G-LTB target gene was detected at 1860 bp (Fig. 1).

3.2. Antigenic analysis of the recombinants

Fluorescence microscopy revealed RV-G-specific immunostain-ing in BHK-21 cells transfected with pVAX1-G, pVAX1-G-LTB andpVAX1-ori-G-LTB (Fig. 2C–E) but not in non-transfected BHK-21cells and BHK-21 cells transfected with the pVAX1 vector (Fig. 2Aand B). The G-LTB protein was predominantly located in the cyto-plasm and retained its antigenic structure.

3.3. Western blot analysis of the recombinants

The lysates of transfected BHK-21 cells were assessed by wes-tern blot analysis to determine the level of expression of the fusiongenes. As shown in Fig. 3, target bands were found in the pVAX1-G-, pVAX1-G-LTB- and pVAX1-ori-G-LTB-transfected cell lysates,whereas no bands were found in the lysates of the non-transfectedBHK-21 cells or the BHK-21 cells transfected with the pVAX1 vec-tor. These results indicate that the three recombinant plasmidssuccessfully expressed their respective antigenic glycoproteins.

3.4. Plasmid stability in vitro

As shown in Fig. 4, the number of recombinant LH430 bacteriacarrying plasmids sharply declined after 12 h and remained stableafter 24 h in vitro.

3.5. Plasmid stability in vivo

The plasmid stability declined in the liver, spleen and PPs at dif-ferent times (Figs. 5A–C).

3.6. IgG induced by the recombinant LH430

The anti-RV IgG serum antibody levels of all groups of mice areshown in Fig. 6. After peaking at days 35 and 45, a statistically sig-nificant difference was observed between the groups of miceimmunised with the recombinant LH430 carrying pVAX1-G-LTBor pVAX1-ori-G-LTB (⁄⁄P < 0.01) and the other immunised groups.These data indicate that LTB vaccination enhanced the humoralimmune response of the immunised mice.

Fig. 5. Plasmid stability after oral infection of mice. Mice were infected with 1 � 1010 CFU LH430 carrying pVAX1, pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The plasmidstabilities in the liver (A), spleen (B), and PPs (C) were assayed. Values represent the average of three mice per data point (mean ± S.D.). The experiment was performed twicewith similar results.

Fig. 6. IgG absorbance at 450 nm of immunised mice. Mice were orally immunisedwith LH430 carrying pVAX1, pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB at days 0and 10. The control group was immunised with PBS on the same days. On days 0, 10,21, 35 or 45, the sera of mice were isolated according to the animal care guidelines,and the levels of anti-RV-G antibodies were analysed by ELISA. ELISA antibody titresare presented as the mean from all mice in each group. Statistically significantdifferences (⁄⁄P < 0.01) were observed among the sera of immunised mice.

Fig. 7. RVNA titres of mice immunised with the recombinant LH430. The barsrepresent the mean RVNA titres in mice immunised with LH430 carrying pVAX1,pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. RVNA titres equivalent to 0.5 IU/mlwere considered to provide adequate protection against rabies, as recommended bythe WHO. Statistically significant differences (⁄⁄P < 0.01, ⁄P < 0.05) were observedamong the sera of immunised mice.

Table 1Survival of mice orally immunised with recombinant attenuated LH430 strains andchallenged with 20 LD50 CVS-11.a

Intracerebrally challengea Survivors/totalb

LH430 (pVAX1-ori-G-LTB) 10 6/10**

LH430 (pVAX1-G-LTB) 10 5/10*

LH430 (pVAX1-G) 10 0/10LH430 (pVAX1) 10 0/10PBS control 10 0/10

a Animals were vaccinated on days 0 and 10 and were challenged with CVS-11 onday 20.

b Number of surviving animals at 14 d after challenge/number of animalschallenged.* P < 0.05 compared with the PBS group.** P < 0.01 compared with the PBS group.

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3.7. Analysis of neutralising antibodies

The mice immunised with recombinant LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB exhibited significantly increased VNAtitres compared with the titres of other immunised groups(⁄⁄p < 0.01). The VNA titres of the mice immunised with recombi-nant LH430 carrying pVAX1-ori-G-LTB were the highest of allgroups (Fig. 7). This study demonstrated that mice immunisedwith recombinant LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB exhibited a VNA immune response that could protectagainst RV challenge (>0.5 IU/ml).

3.8. Protection of the recombinant LH430 immunised mice

Table 1 shows the survival of the mice challenged at with a 20LD50 dose of CVS-11. Sixty and fifty percent of mice immunised

Fig. 8. Serum IL-2 levels in BALB/c mice immunised with recombinant LH430. Atdays 0, 10, 21, 35 or 45, the sera of mice that had been given PBS or LH430 withpVAX1, pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB were collected and analysedby ELISA. Data are expressed as mean values ± S.D. of triplicates. Statisticallysignificant differences (⁄⁄P < 0.01) were observed among the sera of immunisedmice.

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with the recombinant LH430 carrying pVAX1-ori-G-LTB and re-combinant LH430 carrying pVAX1-G-LTB, respectively, were pro-tected against rabies virus challenge (⁄⁄P < 0.01, ⁄P < 0.05). Othergroups of immunised mice unexpectedly succumbed to the chal-lenge. This experiment conclusively showed that protection wasachieved in mice by oral inoculation with recombinant LH430 car-rying pVAX1-G-LTB or pVAX1-ori-G-LTB.

3.9. Cytokine IL-2 ELISA

The IL-2 level may be a key indicator of cellular immune re-sponses. Mice immunised with recombinant LH430 carryingpVAX1-G-LTB or pVAX1-ori-G-LTB had substantially higher IL-2levels than mice immunised with PBS or LH430 carrying pVAX1(⁄⁄P < 0.01) (Fig. 8).

4. Discussion

RV is a negative-stranded RNA virus of the family Rhabdoviridae,genus Lyssavirus (Rath et al., 2005). The RV glycoprotein is a uniqueprotein that induces the production of VNAs that can provide fullprotection against RV challenge (Ertl, 2009). The RV ERA strain oralvaccine based on this glycoprotein has been widely used in foxes,raccoons and other target species (Henderson et al., 2009). Ourstudy will contribute to the development of new oral RV vaccines.

Because of the cellular tropism of Salmonella spp. their rate ofcell-to-cell spread and their dissemination in the body, live atten-uated strains were first used in Salmonella vaccines. The antigensthat Salmonella carries can stimulate humoral and cellular immuneresponses in mice (Loessner et al., 2008; Branger et al., 2010). Re-cently, Salmonella-based vaccines have been widely applied tostimulate immune responses in different animal models (Brangeret al., 2009; Zhang et al., 2010). ST was the first bacterium usedas a recombinant vector for antigen delivery (Curtiss, 2002). Todate, several live recombinant ST-associated vaccines have beenshown to elicit humoral and cell-mediated immunity (Mollenkopfet al., 2001).

A titre greater than 0.5 IU/ml was considered to indicate ade-quate immunogenicity because this titre has been shown to pro-vide full protection against viral challenge, and VNAs are themost protective host response (Rupprecht et al., 2006; Filhoet al., 2011).

LTB vaccination improves antigen uptake by binding with GM1ganglioside on intestinal enterocytes (Wagner et al., 2004;Companjen et al., 2006; Zhang et al., 2009). By contrast, the recom-binant LH430 carrying G-LTB produced higher anti-RV antibodylevels and cellular immune responses in the form of IL-2 produc-tion than recombinant LH430 carrying RV-G.

The plasmid stability in vitro declined sharply because of plasmidloss. Analysis of the plasmid stability showed that the selective pres-sure maintained by the antibiotics was gradually reduced in vitro,causing the stability of plasmid expression in the recombinantstrains to decrease. The plasmid stability of the vector system is alsoan important parameter for vaccination efficiency. The plasmid sta-bility declined throughout the overall observation period. This resultcould be attributed to the metabolic burden within the first sevendays, as the values were determined by plating with an antibiotic.

The serum IgG levels of mice immunised with LH430 carryingG-LTB were significantly elevated in comparison with those inmice immunised with LH430 carrying RV-G. The RV glycoproteinprincipally stimulates the humoral and cellular immune responses.VNAs are a reflection of the immunogenicity of a recombinant pro-tein. In this study, assays for RVNA levels and a CVS-11 challengewere performed to evaluate further the efficacy of LH430 withG-LTB. The data showed that mice immunised with LH430 carryingG-LTB experienced significantly better protection than the otherimmunised mice. Thus, it is presumed that LTB played a crucial rolein the increase in the antibody level.

The higher levels of IL-2 production in mice immunised withrecombinant LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTBindicated that the vaccine induced both Th1 and Th2 responses.RV-specific IgG responses and rabies virus challenge indicated thatthe recombinant LH430-based vaccine carrying pVAX1-G-LTB orpVAX1-ori-G-LTB provided protection against rabies.

5. Conclusions

To produce a highly immunogenic rabies vaccine, we success-fully generated a recombinant live attenuated LH430 strain carry-ing the G-LTB gene. The immune responses induced by and thepotency of the recombinant strains were evaluated using orallyvaccinated mice. The results of the study demonstrated that thisimproved vaccine may have significant clinical implications withrespect to protection against rabies virus infection.

6. Conflict of interest statement

The authors declare that they have no competing interests.

Acknowledgements

This study was supported by the National Major Special Pro-gram for Infectious Disease of China (2008ZX10004-11) and theNational Natural Science Foundation of China (NSFC 30825033,31030064, 30972177, 30950110328, 81070311, 31072147 and31072124).

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