Immunogenic response to a recombinant pseudorabies virus carrying bp26 gene of Brucella melitensis in mice

Research in Veterinary Science 100 (2015) 61–67

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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c

Immunogenic response to a recombinant pseudorabies virus carrying bp26 gene of Brucella melitensis in mice Lan Yao a,1, Chang-Xian Wu a,1, Ke Zheng a, Xian-Jin Xu a, Hui Zhang b, Chuang-Fu Chen b, Zheng-Fei Liu a,* a

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei Province, China Department of Preventive Veterinary Medicine, College of Animal Science & Technology, Shihezi University, Shihezi city, Xinjiang Uyghur Autonomous Region, China b

A R T I C L E

I N F O

Article history: Received 27 October 2014 Accepted 29 March 2015 Keywords: Brucella melitensis Pseudorabies virus bp26 Immunogenicity

A B S T R A C T

Brucellae are facultative intracellular bacterial pathogens of a zoonotic disease called brucellosis. Live attenuated vaccines are utilized for prophylaxis of brucellosis; however, they retain residual virulence to human and/or animals, as well as interfere with diagnosis. In this study, recombinant virus PRV ΔTK/ ΔgE/bp26 was screened and purified. One-step growth curve assay showed that the titer of recombinant virus was comparable to the parent strain. Mice experiments showed the recombinant virus elicited high titer of humoral antibodies against Brucella detected by enzyme-linked immunosorbent assay and against PRV by serum neutralization test. The recombinant virus induced high level of Brucella-specific lymphocyte proliferation response and production of interferon gamma. Collectively, these data suggest that the bivalent virus was capable of inducing both humoral and cellular immunity, and had the potential to be a vaccine candidate to prevent Brucella and/or pseudorabies virus infections. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Members of the Brucella are Gram-negative, non-motile, nonencapsulated, and facultative intracellular bacteria, and cause a zoonotic disease, called brucellosis. They invade professional and non-professional phagocytes, selectively subvert autophagy complexes, and multiply in the endoplasmic reticulum post-traffic in Brucella-containing vacuoles (Roop et al., 2009; Starr et al., 2012). They survive by a “stealth” strategy, in which the low Brucella pathogen-associated molecular patterns are stimulated, and neutrophils are recruited as “trojan Horses” to suppress TH1 response (Barquero-Calvo et al., 2013; Conde-Alvarez et al., 2012). As a consequence, chronic infection establishes. More than half a million new cases are reported annually, and the incidence exceeds 10 human cases per 100,000 population in some countries (Oliveira et al., 2011). Brucella melitensis, B. abortus, and B. suis remain the principal causes of human brucellosis, primarily in Africa, the Middle East and Southeast Asia (Pappas et al., 2006). Humans are infected mainly through contact with infected animals or by contaminated food, and eventually manifest undulant

* Corresponding author. Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. Tel.: +86 27 87644219; fax: +86 27 87282608. E-mail address: [email protected] (Z.-F. Liu). 1 Both authors equally contributed to this work. http://dx.doi.org/10.1016/j.rvsc.2015.03.030 0034-5288/© 2015 Elsevier Ltd. All rights reserved.

fever, arthritis, endocarditis, or other symptoms (Pappas et al., 2005). While no safe and effective human vaccine is available, several vaccines, such as B. suis S2, B. abortus S19, and B. melitensis M5-90, have been developed for disease prevention and control in livestock in some countries (Oliveira et al., 2011; Qiu et al., 2012; Zhong et al., 2013). These vaccines are live vaccines and may occasionally cause abortion in pregnant animals (Baldi et al., 1996; Cloeckaert et al., 2004). Both B. abortus S19 and B. melitensis Rev1 are pathogenic to humans and interfere with diagnosis because they possess the lipopolysaccharide (LPS) containing intact O-chain and induce O-polysaccharide-specific antibodies (Oliveira et al., 2011). DNA vaccines and subunit vaccines, based on outer membrane proteins such as Omp16, Omp31, and BP26 have been widely investigated (Cassataro et al., 2005; Luo et al., 2006; Yang et al., 2005). BP26, also named Omp28 or CP28, is a 26 KDa cytosoluble protein highly conserved in the Brucella genus (Seco-Mediavilla et al., 2003). Recombinant bp26 vaccines have been shown to reduce B. melitensis colonization and to protect against Brucella infection in mice (Yang et al., 2007). Moreover, BP26 was particularly useful for differentiation of infected- and vaccinated- sheep in serological diagnosis (Cloeckaert et al., 1996). Pseudorabies virus (PRV), an alpha herpesvirus, is a cause of the economically important pseudorabies (Aujeszky’s disease) of swine and other livestock (Muller et al., 2011). Live attenuated PRV vaccines have been successfully used to control and eradicate the disease. The genome of PRV is a linear DNA molecule of about 140 kbp, which

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contains several nonessential genes such as thymidine kinase (TK), glycoprotein E (gE), and gC that can be deleted or replaced byheterogeneous genes without affecting the replication of PRV (Szpara et al., 2011) . This makes attenuated PRV a promising candidate for the development of a live vaccine vector to confer protection against both pseudorabies and other diseases. Recombinant PRVΔTK/ ΔgE/LacZ expressing foreign proteins have been assessed as vaccines to protect pigs from PRV and other pathogens (Ju et al., 2005; Li et al., 2008) . In this study, a recombinant pseudorabies virus vaccine strain expressing bp26 of B. melitensis was generated and its immunogenicity was evaluated in mice.

2. Materials and methods 2.1. Bacterial strains, cell lines, virus, and plasmids Competent Escherichia coli strains TOP10 and BL21 (DE3) (Invitrogen) were used for gene cloning and expression, respectively. The B. melitensis vaccine strain M5-90 (usually abbreviated as M5) was originally obtained from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Wang et al., 2011), and grown under aerobic conditions on tryptic soy agar (TSA) or in tryptic soy broth (TSB) as previously described (Zheng et al., 2012). Porcine kidney cell line PK-15, IBRS-2 and bovine kidney cell line MDBK, purchased from China Center for Type Culture Collection (Wuhan), were cultured at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). The live attenuated PRV vaccine strain (ΔTK/ ΔgE/LacZ) was constructed previously (Fig. 1A) and propagated in PK-15. Plasmids pMD18-T (TaKaRa) and pGEX-KG (ATCC77103) were used for gene cloning and expression, respectively. Plasmid pIECMV was constructed previously (Ju et al., 2005), wherein a fragment containing partial gI and gE genes of the pseudorabies virus was deleted, and CMV promoter and BGH poly(A) were incorporated (Fig. 1A).

2.2. Gene cloning and expression M5 genomic DNA was extracted with a genome extraction kit (QIAGEN) and used as template for the amplification of bp26 gene. PCR primers bp26f (5′-GCGGATCCATGAACACTCGTGCTAGCAA-3′) incorporated with a BamH I site, and bp26r (5′-GCGCCATGG TTACTTGATTTCAAAAACGAC-3′) incorporated with a NcoI site were designed to amplify the bp26 gene in full length using DNA polymerase LA Taq (TaKaRa). The PCR product was inserted into pMD18T. The recombinant plasmid carrying bp26 was identified by appropriate restriction enzyme digestion and sequencing. A BamH I-NcoI fragment of 753 bp fragment containing bp26 was subcloned from pMD18-T vector into pGEX-KG. Finally, the recombinant plasmid pGEX-KG-bp26 was electroporated into E. coli BL21. For construction of transfer plasmid, the 753 bp fragment from pMD18-T vector was cleaved and inserted into pIECMV with BamH I and Nco I (Fig. 1B). 2.3. Expression of Brucella BP26 in E. coli The E. coli BL21 harboring the plasmid pGEX-KG-bp26 was activated for average 3 h. Expression of GST-BP26 fusion protein was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture of BL21 cells. The bacteria were harvested and lysed. Lysates were subjected to SDS–PAGE electrophoresis. 2.4. Construction of recombinant virus PRV ΔTK/ΔgE//bp26 The genome of PRV ΔTK/ΔgE/LacZ was digested with EcoR I, and co-transfected into IBRS-2 cells with transfer plasmid pIECMVbp26 by lipofectin reagent (Invitrogen). After cytopathogenic effect (CPE) appeared, the transfected cells were harvested, frozen and thawed three times. Recombinant virus ΔTK/ΔgE//bp26 was screened by plaque purification and PCR using primers bp26f and bp26r. One step growth curve was performed to determine the titer of recombinant virus as per the protocol of Liu et al. (2008). Briefly, cells were infected at a multiplicity of infection (MOI) of 10 at

Fig. 1. Schematic showing construction of the recombinant pseudorabies virus vaccine strain carrying Brucella bp26 gene. A, Genome of the parent vaccine strain PRV ΔTK/ ΔgE/LacZ. Whole genome is composed of unique long (UL), unique short (US), internal repeat (IR) and terminal repeat (TR) regions, including a TK gene deletion in the UL region, gE gene insertion by LacZ. Underneath is the expanded form in which gE locus is inserted by LacZ expression cassette. B, The transfer plasmid pIECMV-bp26 is constructed by inserting a bp26 gene of Brucella into a universal transfer vector pIECMV (Yang et al., 2005), in which partial gI and partial gE of pseudorabies virus are deleted. C, Genome of recombinant virus PRV ΔTK/ΔgE/bp26, in which partial gI and gE gene are replaced by a bp26 gene expression cassette.

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different time points, flasks were harvested, and virus was titrated on MDBK cells. 2.5. Western blot Western blot was performed as described (Liu et al., 2008). Briefly, cellular extracts and purified virion lysates were electrophoresedon a SDS–PAGE gel and transferred to nitrocellulose membrane. Blots were visualized by corresponding antibodies and visualized by 3,3′diaminobenzidine (DAB, Sigma) or luminol staining with SuperSignal WEST Pico kit (Thermo Scientific). 2.6. Animals Four-to-six-week-old female BALB/c mice were purchased from Wuhan Institute of Virology, Hubei province, China; and randomly distributed into experimental groups. Three groups of seronegative mice (each n = 10) were maintained in laboratory isolation cages in Huazhong Agricultural University (HZAU) vivarium throughout the experiments, with food and water freely available. All procedures were approved by the Hubei Province Animal Care and Use Committee. 2.7. Preparation of polyclonal antibody The purification of BP26 was carried out by a GST purification kit (Shanghai Sangon Co., Ltd). To make hyperimmune serums, the GST-BP26 fusion protein purified by binding to glutathione– sepharose beads was used to immunize mice by standard procedure. After three times of booster immunization every 2 weeks, mice were bled and hyperimmune serum was isolated. 2.8. Immunization of recombinant PRV ΔTK/ΔgE/bp26 virus in BALB/c mice Four-week-old female BALB/c mice (30) were equally divided into three groups (each n = 10), group 1 and group 2 were inoculated with 100 μl of 1 × 105 TCID50/ml of PRV ΔTK/ΔgE/bp26 and PRV ΔTK/ ΔgE/LacZ, respectively, and control group with 100 μl DMEM. All mice were immunized intramuscularly in the hind legs and a similar booster dose given 14 days later. The mice were bled from tail and serum was obtained at 1000 × g for 10 min at 4 °C. 2.9. Enzyme-linked immunosorbent assay (ELISA) ELISA was performed as described (Yang et al., 2007). Briefly, a 96-well plate was coated with BP26 protein, blocked with 5% bovine serum albumin (BSA) solution, and incubated with the mice serum in 1:1600 dilution at 37 °C for 1 h. Subsequently, HRP-conjugated goat anti-mouse IgG in 1:5000 dilution was added to each well, and visualized with 3,3′,5,5′-tetramethylbenzidine (TMB). 2.10. Serum neutralization test Serum neutralization assay was performed as previously described (Ju et al., 2005). Briefly, mice serum was two-fold serially diluted in DMEM, and incubated with 100 TCID50 of PRVΔTK/ΔgE/ LacZ at 37 °C for 1 h. Then IBRS-2 cells were added to each well and incubated at 37 °C with 5% CO2 for 4–6 days until CPE appeared. Appropriate serum, virus and cell controls were included in the tests. Neutralization titer were calculated as the reciprocal of the highest dilution resulting in complete neutralization with Reed–Muench quantal assay (Varedi et al., 2014) .

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2.11. Lymphocyte proliferation assay Lymphocyte proliferation response assay was performed as described earlier (Ju et al., 2005). Briefly, spleens were dissected from the immunized mice and were homogenized in PBS (pH 7.4). The erythrocyte suspension was lysed with 0.75% Tris–NH4Cl (pH 7.4). After washing three times with PBS, the splenocytes were resuspended in RPMI-1640 supplemented with 10% FBS, 14 mM HEPES, 50 mM 2-mercaptoethanol, 100 mg/ml streptomycin and 100 IU/ml penicillin. Splenocytes were seeded in 96-well plate at 100 μl per well (4 × 106 cells per well) in triplicate. Meanwhile, Brucella M5 was subjected to UV inactivation at a wavelength of 250 nm for 30 min. The viability of inactivated bacteria was tested on TSA plate. Subsequently, 100 μl per well of medium with or without UVinactivated Brucella M5 at 5 or 10 μg/ml was added and mixed, and incubated for 72 h in 37 °C with 5% CO2. The 20 μl of MTS (3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, Promega) was added to each well. After further incubation for 4–6 h, the plates were read at OD492 nm. Stimulation index was calculated as the ratio of average OD492 value of M5/average OD492 value of negative control. 2.12. Cytokine ELISPOT For detection of IFN-γ, splenic lymphocytes were stimulated by the UV-inactivated Brucella M5 (10 μg/ml) and incubated for 72 h at 37 °C. IFN-γ level in culture supernatants was measured by an ELISA Quantikine Mouse kit (PharMingen). 2.13. Statistical analysis Student’s t test was used for statistical analysis. P-value <0.05 is considered statistically different, and P values <0.01 are considered significantly different. 3. Results 3.1. Expression of Brucella BP26 protein in E. coli According to B. melitensis 16M strain genome sequence (GenBank accession number: NC_003317.1), bp26 gene was amplified by PCR using primer pair bp26f and bp26r. A 753 bp specific fragment, from 557729 to 558481 in the complementary strand of chromosome I, was cloned and sent for sequencing. Sequence analysis showed that bp26 of Brucella M5 is 100% identical with B. melitensis 16M strain (data not shown). To identify the Brucella BP26 protein, bp26 gene was cloned and fused to the downstream of GST coding sequence, and transformed in E. coli BL21. After the induction by IPTG, E. coli was lysed and subjected to SDS–PAGE electrophoresis. The apparent molecular mass of BP26 polypeptide was estimated to be about 26 KDa, and fusion protein was about 54 KDa. The best condition for expression was induction with 1 mM IPTG at 37 °C for 4 h. The expressed fusion protein was soluble in the supernatant of bacterial lysate (Fig. 2A). Immunoblot assay showed that the fused GSTBP26 protein was about 54 KDa, which was recognized by goat antiBrucella positive antibody (Fig. 2B). 3.2. Brucella bp26 gene was incorporated and expressed in recombinant virus After cotransfection and plaque purification, recombinant virus was identified by two PCRs. The first PCR was conducted using bp26f and bp26r, and the second PCR using LacZf and LacZr as

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Fig. 2. Expression of Brucella bp26 gene in E. coli. A, Brucella bp26 gene was cloned by PCR and inserted into pGEX-KG vector, the resulting plasmid pGEX-KG-bp26 was transformed into E. coli BL21. Bacteria were induced by IPTG and subjected to SDS–PAGE electrophoresis. Lanes 1–6 are the bacterial induced with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mM IPTG, respectively. Lane M, Prestained protein marker. Lane 7, pGEX-KG-bp26 vector control without IPTG induction, and lane 8, pGEX-KG vector control induced with 1.0 mM IPTG. B, Western blot assay. BP26 protein in SDS–PAGE gel was transferred onto nitrocellulose membrane, incubated with goat anti-B. melitensis positive serum (1:50) overnight, followed with HRP-conjugated rabbit anti-goat IgG (Thermo) (1:5000) for 4 h, and visualized by the substrate DAB (Thermo Scientific). Lane 1, pGEX-KG control. Lane 2, pGEX-KG-bp26. Arrowheads indicate the predicted bands.

Fig. 3. Identification of the recombinant virus PRV ΔTK/ΔgE/bp26. A, Identification of bp26 gene in recombinant virus by PCR with the primers bp26f and bp26r. Lane M, DNA marker. Lanes 1–12, Different plaques of recombinant virus. B, Detection of LacZ gene in recombinant virus and the parent viruses by PCR using primers LacZf (GAACTGCCTGAACTACC) and LacZr (ACTGCAACAACGCTGC) (Liu et al., 2002). Lane 1, Parent virus PRV ΔTK/ΔgE/LacZ. Lane 2 and lane 3, recombinant virus PRV ΔTK/ΔgE/ bp26. C, Western blot assay showing the expression of bp26 gene in recombinant virus. PK-15 cells were infected with either parent or recombinant viruses at an MOI of 10, cells were harvested when 80% CPE appeared, and cell lysates were prepared and subjected to SDS–PAGE electrophoresis. Polyclonal antibody against BP26 made in mice (1:100) and HRP-conjugated goat anti-mouse IgG (Thermo) (1:5000) were used as first and secondary antibodies. The blot was incubated with first and secondary antibodies for overnight and 4 h, respectively. Finally, the blot was visualized by SuperSignal WEST Pico kit (Thermo Scientific). Lane 1, Recombinant virus. Lane 2, Parent virus. Lane 3, Mock control.

primers (Liu et al., 2002). A specific 753 bp fragment containing bp26 gene was detected from all recombinant plaques, but was absent in the control (Fig. 3A). In contrast, the specific 490 bp of LacZ gene was present in the parent strain, and absent in the recombinant virus (Fig. 4B). The result indicated that bp26 was incorporated properly into the genome, where the LacZ gene was replaced. To clarify the expression of bp26 gene in the recombinant virus, PK-15 cells were infected by the recombinant virus, parent virus, or mock at 10 MOI, and cells were harvested when 80% CPE appeared. Cells were lysed and subjected to SDS–PAGE, followed by blot onto nitrocellulose membrane. The polyclonal antibody against BP26 made in mice was used as first antibody, HRP-conjugated goat anti-mouse IgG was used as secondary antibody, and visualized in the presence of DAB. A specific band of about 26 KDa appeared as expected for the recombinant virus, but was absent for the parent virus control (Fig. 3C). This result indicated that bp26 was correctly expressed in recombinant virus-infected cells.

Fig. 4. Replication property of the recombinant PRV carrying Brucella bp26 gene. One-step growth curve was performed as described earlier (Liu et al., 2008). Briefly, PK-15 cells were infected either by recombinant virus PRV ΔTK/ΔgE/bp26 or by the parent virus PRV ΔTK/ΔgE/LacZ at an MOI of 10. Cells were harvested at indicated time points, and titrated on MDBK cells.

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3.3. The recombinant virus grew to a similar titer compared with the parent virus To address whether the insertion of Brucella bp26 affected the replication of PRV, one-step growth curve was conducted. The multiplication of recombinant PRV was similar to parent virus at 0, 3, 6, 12, 18, 24, and 30 h post-infection. This indicated that insertion and expression of bp26 gene did not affect the replication of PRV. 3.4. Recombinant virus was able to stimulate high level of humoral immune response Throughout the period of animal experiment, all immunized mice were as healthy as the mock-inoculated controls. A previous study showed that immunization with purified bp26 and conjunction with cholera toxin (CT) adjuvant elicited systemic immune responses (Yang et al., 2007). To test the serum antibody against BP26, 4–6 week old mice were immunized with either recombinant virus PRV ΔTK/ΔgE/ bp26, or parent virus PRV ΔTK/ΔgE/LacZ, or mock-injected and boosted 2 w after the primary immunization. Serum was obtained weekly and the titers of antibody against BP26 were determined by ELISA. Compared to the negative control and pre-immunization sera, neither recombinant virus nor parent virus elicited enough antibody in the first and second weeks post-vaccination. However, at 4 and 6 weeks post-vaccinations, there was a significant difference in antibody titer (P < 0.01), and recombinant virus elicited two-fold titer more than parent virus and mock-vaccinated group (Fig. 5A). To determine the antibody against PRV, a serum neutralization test was performed. The serum from immunized mice was incubated with virus, and the suspension was mixed and cultured until CPE appeared. At 4 w post-vaccination, low to no neutralization antibody was detected in all three groups. However, at 6 w postvaccination, both recombinant virus and parent virus elicited high neutralization antibody titer, but there was no difference among those groups (Fig. 5B). These data demonstrate that the recombinant virus carrying bp26 of Brucella was able to elicit good humoral immune response. 3.5. Recombinant virus was capable of stimulating high level of cellmediated immune response At 4 w and 6 w post-primary vaccination, mice were sacrificed for splenocyte proliferation response. The Brucella specific splenocyte

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proliferation response was significantly higher in the group 1 than group 2 and control group at 6 w post-primary vaccination (P < 0.01), but there was no difference at 4 w. The most effective dose of M590 was 5 μg/ ml, and the stimulation indexes of recombinant virus, parent virus, and DMEM control were 1.91, 1.21, and 1.14, respectively (Fig. 6A). To further investigate the cell-mediated immune response, a ELISPOT was performed to examine the IFN-γ production level. As shown in Fig. 6B, IFN-γ increased in 6 w compared to 4 w, and the titer stimulated by recombinant virus was significantly higher than parent and control group (P < 0.01). The titers of IFN-γ stimulated by recombinant virus, parent virus, and DMEM are 148.37, 72.31 and 69.61 pg/ml, respectively (Fig. 6B). These data suggested that the recombinant virus PRV ΔTK/ΔgE/bp26 was capable of inducing good cell-mediated immune response. 4. Discussion Brucellosis is one of the most common zoonoses in the world, and no safe and effective human and/or animal vaccines are available (Perkins et al., 2010). Currently, a few live attenuated vaccines are used in some countries, but they have residual virulence and side effects (Oliveira et al., 2011). There is an urgent need to develop new vaccines that are safe and efficacious. To the best of our knowledge, this is the first report about the expression and immunity of an intracellular bacterium-origin gene in pseudorabies virus vaccine vector. Although the natural hosts of PRV are swine and wild boars, the virus is infective and fatal to most livestock. Live marker vaccine, in which gE and TK genes are deleted, is extensively used to control and eradicate the associated disease in pigs. Its multiple-species tropism makes PRV vaccine virus one of the best vectors to develop bivalent or trivalent vaccines (Hong et al., 2007; Ju et al., 2005; Li et al., 2008; Xu et al., 2004). In fact, multiple foreign genes from immunological proteins of viruses were inserted and expressed in PRV. LacZ, a bacterium-origin gene, was also successfully expressed in PRV (Liu et al., 2002). BP26, an outer membrane protein, is very conservative and immunological in all Brucella species (Yang et al., 2007). In our study, sequence analysis of Brucella M5 bp26 indicated that it was 100% identical with reference strain B. melitensis 16M (data not shown). Western blot showed that this protein was expressed in appropriate molecular weight, as expected in E. coli and in recombinant

Fig. 5. Humoral immunity of mice elicited by the recombinant PRV carrying Brucella bp26 gene. Mice were immunized with recombinant virus PRV ΔTK/ΔgE/bp26, the parent virus PRV ΔTK/ΔgE/LacZ, or with DMEM, and boostered at 2 weeks post-primary injection. Serum was subjected to ELISA for detection of antibody against Brucella BP26 (A), or subjected to serum neutralization test for detection of antibody against PRV (B).

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Fig. 6. Cellular immunity of mice elicited by the recombinant PRV carrying Brucella bp26 gene. Lymphocytes were isolated from the spleens of immunized mice, and stimulated by UV-inactivated Brucella M5, lymphocyte proliferation and IFN-γ was determined by lymphocyte proliferation assay (A), and cytokine ELISPOT assay (B), respectively. Detailed protocol for the above two assays were referred to the manufacturer’s instructions or described in Section 2.

virus-infected cells (Figs. 2B and 3C). The results suggested that (i) bp26 gene was inserted and expressed correctly, and (ii) insertion and expression of bp26 did not affect the infection and replication of PRV. Brucella spp. are facultative intracellular bacteria, and humoral and cellular immunities are important in their elimination (Barrionuevo et al., 2013; Starr et al., 2012). In animal experiments, the mice vaccinated with recombinant virus produced higher titer of antibodies against BP26 at 6-weeks post-primary and 4 week post-booster immunizations (Fig. 5A). Both viruses stimulated high titer of neutralization antibody against PRV, and there were no differences between the recombinant and parent virusvaccinated mice (Fig. 5B). Meanwhile, as indicated in Fig. 6, the spleens of mice vaccinated with recombinant virus had stronger lymphocyte proliferative response and higher level of IFN-γ than the other two groups. IFN-γ is the characteristic cytokine of Th1 immune response, while the Th1 immune response characterized by IFN-γ is associated with protection against brucellosis (Eze et al., 2000; Murphy et al., 2001; Zhan and Cheers, 1993). It is noticeable that both humoral and cellular immune responses were low at 4 w post-primary vaccination, or at 2 w post-booster, but increased to high levels at 6 w post-primary vaccination, or 4 w post-booster. We speculated that this phenomenon might be due to the neutralization effect of the existing immunity of mice to the live virus booster, which is in agreement with our previous observation for another recombinant virus (Ju et al., 2005). Protection efficacy of the recombinant virus as well as the experiment to determine bacterial load of Brucella post-challenge are in progress. Based on the protective ability of BP26 in BALB/c mice vaccinated with bp26 DNA vaccines (pcDNA3.1) showing reduced splenic colonization post-challenge (Yang et al., 2005, 2007), we believe that this recombinant virus might confer protection against challenge by Brucella and/or PRV. In conclusion, we described a recombinant PRV that expresses Brucella BP26 protein using PRV live vaccine strain as a vector. The virus induced both humoral and cellular responses with high titer. The data suggest that this recombinant virus may be a promising vaccine candidate for both Brucella and PRV infection in livestock. Acknowledgements This work was supported partially by Natural Science Foundation of China (31270293) to Z.-F. Liu, partially by 973 Plan (2010CB530203) to Z.-F. Liu and C.-F. Chen, and the Fundamental Research Funds for Central Universities (2014PY038) to Z.-F. Liu.

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