Microbial Pathogenesis 69-70 (2014) 53e59

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Recombinant BCG coexpressing Ag85B, ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice Enzhuo Yang a,1, Yunzhi Lu a,1, Ying Xu a, Qing Liang b, Chao Wang a, Honghai Wang a, *, Hongbo Shen a, * a b

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China Rizhao Polytechnic, Rizhao, Shandong 200433, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 July 2012 Received in revised form 27 March 2014 Accepted 31 March 2014 Available online 12 April 2014

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4þ and CD8þ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-g, TNF-a and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Mycobacterium tuberculosis rBCG Th1 Cytokines Vaccine

1. Introduction Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (MTB), is one of the major health problems in the world and responsible for 1.3 million deaths annually [1]. The emergence of multidrug-resistant TB (MDR-TB) and human immunodeficiency virus (HIV) infection aggravates this problem [2]. Currently, Bacillus Calmette-Guérin (BCG) is the only tuberculosis vaccine available for human [3] and has been given to more than 3 billion people since 1921 [4]. Although being the world’s most widely used vaccine, BCG is also the most controversial vaccine in use [4,5]. It provides good protection against severe forms of childhood TB such as miliary and meningitis tuberculosis. However, its protective efficacy against adult tuberculosis in endemic areas is insufficient, ranging from 0 to 80% [6]. Therefore, a more effective vaccine against MTB is in urgent need.

* Corresponding authors. Tel.: þ86 21 6564 3777; fax: þ86 21 6564 8376. E-mail addresses: [email protected] (H. Wang), [email protected] (H. Shen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2014.03.011 0882-4010/Ó 2014 Elsevier Ltd. All rights reserved.

A great number of efforts have been made to develop new vaccines against TB. One opinion is that some antigens are missing during BCG passage. As a result, genetic modification of BCG is one promising approach to increase its immunogenicity. The strategies for recombinant BCG are usually as follows: to recombinant missing genes back into BCG; to increase the expression level of immunodominant genes. In conclusion, an effective vaccine requires for the ability to elicit protective immune response. Multigenic vaccine strategy is attractive because of the ability to generate broader immune responses by simultaneously covering several antigens. Antigen 85B (Ag85B) is one of the major secreted proteins observed in MTB or BCG culture filtrates [7]. rBCG vaccines overexpressing Ag85B could provide better protective efficacy than BCG against aerosol challenge of MTB in guinea pig [8,9]. Early secreted antigenic target of 6 KDa (ESAT-6) is considered to be a dominant antigen for cell-mediated immunity [10] and a major target for memory T cells in mice infected with MTB [11]. Rv3620c, a member of the ESAT-6 family, is a secretory protein reported in culture filtrates of MTB [12]. Rv3620c is expressed in MTB, whereas no expression of Rv3620c was detected in Mycobacterium bovis and attenuated strain H37Ra [13,14]. Evidence

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suggests that ESAT-6 family is served as a source of T-cell antigens, which can be exploited for the development of vaccines against MTB [15]. Rv3620c stimulation could trigger Th1 immune response and an increased production of IFN-g in peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative of tuberculin positive (PPDþ) donors [16]. In the present study, we have successfully constructed a recombinant BCG expressing Ag85B-ESAT6-Rv3620c fusion protein. Our data supported the conclusion that a single immunization of this rBCG::Ag85B-ESAT6-Rv3620c triggered a more efficient immune response than BCG. And it may serve to be a promising candidate vaccine against MTB for further study. 2. Results 2.1. Construction and identification of rBCG expressing Ag85BESAT6-Rv3620c fusion protein To construct this rBCG, the coding regions of ag85b, esat-6 and rv3620c were cloned into the shuttle vector pMV261 to form a recombinant vector. After identified by DNA sequencing, this recombinant vector was transformed into BCG Danish Strain to generate recombinant BCG. rBCG clones harboring recombinant vector were cultured in 7H9 medium and heat-shocked for 2 h. Then the supernatant was subjected to Western blotting. An antiAg85B rabbit polyclonal antibody was employed to test the expression level of fusion protein and BCG strain was regarded as control. It was observed that rBCG successfully expressed the fusion protein with a molecular mass of 57.3 KDa (Fig. 1). 2.2. Antigen-specific humoral response induced by rBCG immunization In this study, to evaluate antibody response induced by rBCG, we examined the Ag85B-specific antibody titres of IgG, IgG1 and IgG2b [21] in mice serum at 4th and 8th week after immunization. Serum from each mouse was subjected to ELISA to test the antibody titer against Ag85B protein. There was no difference between BCG and rBCG at 4th week after vaccination. However, 4 weeks later at 8th week, a significantly elevated IgG titer was found in rBCG-immunized mice (Fig. 2a). This result indicates that rBCG could induce more efficient humoral response than BCG at a much longer time schedule. Humoral response is essential for TB protection. And cellular response is much more important for the reason that MTB is

identified to be an intracellular pathogen. It is reported that Th1 immune response contributes to TB control. So we determined the titers of IgG1 and IgG2b against Ag85B to evaluate the immune response. No difference was found at 4th week after vaccination. However, at 8th week, the IgG2b/IgG1 ratio in rBCG-immunized group (3.7  0.7) was much higher than that in BCG-immunized group (1.6  0.3) (P < 0.05) (Fig. 2b). The results revealed that rBCG could induce a significantly strong humoral response and improve the Th1 immune response in C57BL/6 mice after immunization. 2.3. rBCG immunization induced antigen-specific Th1 immune response To confirm the conclusion that rBCG could induce Th1 immune response. We measured the expression level of IFN-g. IFN-g is an important Th1 type cytokine that activates macrophages and promotes Th1 immune response [22]. Here, we separated spleen lymphocytes from mice immunized with rBCG or BCG at 8th week after vaccination. Then spleen lymphocytes were stimulated with Ag85B or PPD for 30 h and supernatant was tested for the concentration of IFN-g by ELISA. As expected, a much higher expression level of IFN-g was found in mice immunized with rBCG. Both Ag85B and PPD could induce a higher concentration of IFN-g in rBCG-immunized mice than those immunized with BCG (Fig. 3). The results demonstrated that rBCG could induce more IFN-g production than BCG and enhance the immunogenicity in mice. TNF-a was also suggested to play an important role in TB control [23,24]. So we have evaluated the response of TNF-a at 8th week after rBCG vaccination. 2.5  105 spleen lymphocytes were stimulated with Ag85B or PPD for 30 h and the number of TNF-a secreting cells was tested by ELISPOT. It was noted that after stimulation, there were more spleen lymphocytes releasing TNF-a from mice immunized with rBCG than those immunized with BCG (P < 0.05) (Fig. 4). As a result, rBCG exhibited a stronger immunogenicity than the traditional BCG vaccine since it could induce more TNF-a secreting than BCG. IL-2 is a Th1 cytokine, which may elicit a Th1 immune response early in response to vaccination [25]. To further characterize immune responses by immunization with rBCG and BCG, the expression level of IL-2 at 8th week was detected by ELISA in culture supernatant of spleen lymphocytes stimulated with antigens Ag85B or PPD in vitro, respectively. Coincidentally, compared with BCG vaccinated mice, a higher level of IL-2 was found in the culture supernatants of spleen lymphocytes from rBCG-immunized mice (Fig. 5). These results demonstrated that rBCG immunization could trigger Th1 immune response. Then the concentration of IL-10 was measured in supernatant. IL-10 is usually associated with Th2 immune response. Although there was no difference between rBCG-immunized group and BCGimmunized group after Ag85B stimulation. There was a significantly decrease in IL-10 secretion in rBCG vaccinated group after PPD stimulation (Fig. 6). Above all, these results demonstrate that rBCG immunization could induce an enhanced Th1 immune response and suppress Th2 immune response, which is beneficial for TB control. 2.4. rBCG vaccination induced CD4þ and CD8þ lymphocytes proliferation

Fig. 1. Western blotting analysis of recombinant BCG. rBCG or BCG was cultured in 7H9 medium and heated shock at 45  C for 2 h. Supernatants were collected and analyzed for expression of recombinant antigens Ag85B-ESAT6-Rv3620c by using anti-Ag85B rabbit polyclonal anti-sera. Lane 1: BCG; Lane 2: rBCG; M: Protein marker.

Previous studies have shown that both CD4þ and CD8þ T lymphocytes play an active role in inducing specific immune response to M. tuberculosis antigens [26e28]. In this study, the proliferation of both CD4þ and CD8þ T lymphocytes was analyzed in vitro at 8th week. Spleen lymphocytes were separated and co-cultured with

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Fig. 2. Antibody response against specific antigen Ag85B in immunized mice. C57BL/6 mice were immunized with BCG or rBCG. After 4 or 8 weeks, mice were sacrificed and sera was obtained and subjected to ELISA. Ag85B (1mg/well) was used for detecting IgG, IgG2b and IgG1 titer by ELISA assay. (a) rBCG immunization reveals an increased IgG response to Ag85B; (b) rBCG vaccination exhibits a much higher ratio of IgG2b/IgG1 than BCG. Data was depicted as mean values SEM, and the statistical significance was determined by Student’s t-test (*, P < 0.05).

Ag85B or PPD for 30 h. Then the cell pellets were stained by fluorescein antibodies and subjected to flow cytometry to determine the proportion of CD4þ and CD8þ T cell. No significant difference was observed after Ag85B stimulation. However, after PPD stimulation, the proportion of CD4þ T cell subset in rBCG-immunized mice (35.9  1.5%) is much higher than that in BCG-immunized mice (29.6  2.0%) (P < 0.05) (Fig. 7a). Also, the percentage of CD8þ T cell subset in spleen lymphocytes of rBCG-immunized mice (24.1  1.4%) is significantly higher than that in BCG-immunized mice (16.9  1.0%) (P < 0.05) (Fig. 7b). The ratio of CD8þ/CD4þ T

Fig. 3. IFN-g production in vitro by spleen lymphocytes of immunized mice. Mice immunized were sacrificed at 8th week. Spleen lymphocytes were isolated aseptically and plated in 24-well plates in 2 ml RPMI-1640 medium per well containing PPD (2 mg/ ml) or Ag85B (2 mg/ml) protein, respectively. After incubating for 30 h at 37  C, 5% CO2, the supernatants were tested by ELISA assay. Values of IFN-g production were expressed as mean (SEM), and the statistically significance was performed by Student’s t-test (*, P < 0.05).

cell was calculated to determine the proliferation efficiency. It was found that rBCG-immunized mice revealed a CD8þ/CD4þ ratio of 0.70  0.03, which is much higher than those immunized with BCG (0.57  0.02) (Fig. 7c). Our data demonstrated that both CD4þ T cells and CD8þ T cells could be induced by rBCG immunization. The proliferative response of CD8þ T cells was stronger than CD4þ T cells. These results demonstrated that rBCG could induce significantly stronger immune responses, especially cytotoxic effect in mice.

Fig. 4. Frequency of TNF-a-secreting cells in spleen lymphocytes of immunized mice. Mice immunized were sacrificed at 8th week for spleen lymphocytes separation. The isolated spleen lymphocytes (1.25  106 cells/ml) were stimulated in 96-well ELISPOT plates with PPD (2 mg/ml), Ag85B (2 mg/ml or 4 mg/ml) or PHA (2 mg/ml), respectively. The plates were incubated for 30 h at 37  C, 5% CO2. ELISPOT essay was performed to evaluate the number of TNF-a-secreting cells. Data were presented as mean values SEM, and the difference was statistically significance using Student’s t-test (*, P < 0.05).

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Fig. 5. IL-2 production in vitro by splenocytes of immunized mice, following stimulation with PPD or Ag85B protein, respectively. Mice immunized were sacrificed at 8th week. Spleen lymphocytes were cultured at a concentration of 1  106 cells/ml in 2 ml volume with PPD (2 mg/ml) or Ag85B (2 mg/ml) protein, respectively. The supernatants were tested by ELISA assay after 30 h incubation at 37  C, 5% CO2. Values of IL-2 production were expressed as mean (SEM), and the statistically significance was performed by Student’s t-test (*, P < 0.05).

3. Discussion Although the current TB vaccine BCG has been widely used, its efficacy is highly inconsistent in several well-controlled clinical trials [29]. The development of novel vaccines against M. tuberculosis is in urgent need to facilitate worldwide control of tuberculosis. Multigenic vaccine strategy is an attractive way by expressing a variety of different protective epitopes and generating broader immune responses [23]. Based on this strategy, we constructed a new recombinant BCG expressing multiple antigens and evaluated its immune response in mice.

Both humoral and cellular immune response is evaluated in mice immunized with this rBCG. Compared with BCG-immunized group, rBCG immunization could generate significantly higher antibody titers. Two main types of the T lymphocytes, both CD4þ and CD8þ T cells are crucial for protective immunity against M. tuberculosis [30]. CD4þ T cells could produce cytokines to activate macrophages and CD8þ T cells take charge in lysing infected cells and eliminating M. tuberculosis [31,32]. In our study, both CD4þ and CD8þ T cells from rBCG vaccinated mice could be induced after stimulated with PPD. Moreover, there was more CD8þ T cells proliferation than CD4þ T cells in rBCG-immunized mice, which indicate that rBCG triggered a stronger immune response. Previous evidence shows that the role of Th1 and Th2 immune response during M. tuberculosis infection is different. Th1 immune response triggers cell-mediated immunity and anti-intracellular pathogen responses while Th2 immune response stimulates predominantly anti-extracellular pathogen humoral responses. Th1 immune response could contribute to M. tuberculosis elimination. In contrast, Th2 immune response is associated with progressive infection of M. tuberculosis [33]. Here, we determined the expression level of IFN-g, TNF-a, IL-2 and IL-10 in culture supernatant, respectively. The results showed that spleen lymphocytes from rBCG-immunized mice produced more IFN-g, TNF-a and IL-2 cytokine than those from BCG-immunized group, whereas the secretion level of IL-10 from rBCG-immunized mice was lower. IFNg, TNF-a and IL-2 are typical Th1 cytokines, while IL-10 is a typical Th2 cytokine. Here, the results revealed that rBCG could effectively elicit cell-mediated immune response by inducing more Th1 cytokines and suppressing Th2 immune response [34]. All the immune responses are necessary for protection against M. tuberculosis [35]. Our findings show that a more robust Th1 immune response was generated by this novel rBCG vaccine. Th1 immune response is characterized by a strong cellmediated immunity and an IgG2b antibody response, whereas Th2 immune response is usually associated with high titer of IgG1 and IgE antibody response [36]. To verify our conclusion, the levels of Ag85B-specific IgG1 and IgG2b were tested in sera of mice. The results showed that the ratio of IgG2b/IgG1 is significantly higher in rBCG-immunized mice than that in BCG-immunized mice. Once again, it demonstrated that rBCG could induce stronger Th1 responses than BCG in mice. The results from this study indicate that rBCG::Ag85B-ESAT6Rv3620c could induce a stronger Th1 immune response as well as a much stronger humoral response than traditional BCG in mice. This rBCG may serve to be a new vaccine candidate for TB protection. 4. Materials and methods 4.1. Animals 6-8 weeks-old female C57BL/6 mice were purchased from Slaccas Inc. (Shanghai, China). Animal experiments were performed in accordance with the guidelines of the Chinese Council on Animal Care. All mice were maintained under specific-pathogen-free conditions throughout the experiments. 4.2. Bacterial strains and media

Fig. 6. IL-10 production in vitro by splenocytes of immunized mice, following stimulation with PPD or Ag85B protein respectively. Mice immunized were sacrificed at 8th week. Spleen lymphocytes were cultured at a concentration of 1  106 cells/ml in 2 ml volume with PPD (2 mg/ml) or Ag85B (2 mg/ml) protein, respectively. The supernatants were tested by ELISA assay after 30 h incubation at 37  C, 5% CO2. Values of IL-10 production were expressed as mean (SEM), and the statistically significance was performed by Student’s t-test (*, P < 0.05).

BCG (obtained from Shanghai Biological Products Institute) and rBCG were cultured in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI) supplemented with 0.5% glycerol, 0.05% Tween80 and 10% ADC or solid Middlebrook 7H10 medium (Difco Laboratories) supplemented with ADC. When required, the antibiotic kanamycin was added at a concentration of 25 mg/ml. E. coli DH5a

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(TransGen Biotech, Inc.) used for cloning was grown in LuriaBertani medium at 37  C, supplemented with either 100 mg/ml ampicillin or 25 mg/ml kanamycin, according to the resistant gene present on the plasmid. Antigen 85B recombinant protein without signal peptide was purified by affinity chromatography with Ni2þ column. PPD was obtained from Shanghai Biological Products Institute. 4.3. Construction of rBCG The coding sequence for ag85b and esat-6 were cloned from MTB strain H37Rv genome DNA by PCR methods. Then the two gene fragments were cloned into shuttle plasmid pMV261 to generate recombinant shuttle plasmid pMV261-ag85b-esat6 (pMV261-AE) [17]. The gene fragment rv3620c was amplified by PCR from H37Rv genome. The forward primer was 50 -AAT GTC GAC ATG ACC TCG CGT TTT ATG AC-30 (Sal I sit underlined) and the reverse primer was 50 -AAT GTT AAC TCA GCT GCT GAG GAT CTG C-30 (Hpa I sit underlined). The resulting 297bp fragment was ligated into the T-Cloning Site of pMD19-T vector (TaKaRa Inc.). After digesting with Sal I and Hpa I, the gene fragment was ligated into the digested recombinant shuttle plasmid pMV261-AE to generate pMV261-AE-rv3620c. Fidelity of the construction was confirmed by DNA sequencing. The recombinant shuttle plasmid was transformed into BCG Danish strain by electroporation as described previously [17]. The transformed BCG strains were incubated on 7H10 plates supplemented with 25 mg/ml kanamycin at 37  C for 2e3 weeks. Transformants were identified and cultured in Middlebrook 7H9 medium containing 25 mg/ml kanamycin. After 2e3 weeks’ growth, the protein expression was induced by heating at 45  C for 2 h [18]. rBCG cell pellets were centrifuged at 8,000 g for 20min and culture supernatants were concentrated as described previously [18]. Supernatants were analyzed for expression of recombinant antigens by Western blotting. 4.4. Immunization C57BL/6 mice were immunized subcutaneous with recombinant BCG (rBCG) or BCG Danish Strain (BCG) at a dose of 4  106 CFU. At 8th week after immunization, mice were sacrificed to analyze immune response. 4.5. Cytokine production The immunized mice were sacrificed at 8th week after immunization. Spleens were removed aseptically and gently grinded in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 mM b-mercaptoethanol, 100 mg/ml streptomycin and 100U/ml penicillin. Spleen lymphocytes were prepared by densitygradient centrifugation with Lympholyte-M (Cedar Lane Lab, Burlington, NC, USA). Then the cells were adjusted to a concentration of 1  106 cells/ml and grown in 24-well plates (G&H) in RPMI-1640 medium as described above. The concentration of either stimulus added was as follows: 2 mg/ml PPD or 2 mg/ml Ag85B. The final volume per well was 2 ml. The cells were incubated at 37  C for 30 h

Fig. 7. Proportion of CD4þ and CD8þ T cell subsets and the ratio of CD8þ/CD4þ T cells, obtained from the spleen of immunized mice. Mice immunized were sacrificed at 8th week. Spleen lymphocytes were cultured at a concentration of 1  106 cells/ml in 2 ml

volume with PPD (2 mg/ml) or Ag85B (2 mg/ml) protein, respectively. After 30 h incubation at 37  C, 5% CO2, the lymphocytes were harvested and CD4þ and CD8þ T cell subsets were double-stained for FACS-analysis using FITC-labeled CD4 mAb and PElabeled CD8 mAb. (a) The percentage of CD4þ T cell subsets after stimulation. (b) The percentage of CD8þ T cell subsets after stimulation. (c) The ratio of CD8þ/CD4þ T cells after stimulation. The mean percentage values of CD4þ T cells and CD8þ T cells, and the ratio of CD8þ/CD4þ T cells were presented as mean (SEM), and the statistically significant differences were revealed using Student’s t-test (*, P < 0.05).

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in a humidified CO2 incubator. Supernatants from each well were collected and frozen at 20  C for ELISA assay.

Software Foundation, USA). P < 0.05 was considered statistically significant.

4.6. ELISPOT assay for TNF-a

Acknowledgments

Mice were sacrificed at 8th week after immunization and spleen lymphocytes were separated as described above. Mouse TNF-a ELISPOT kit (U-Cytech biosciences, CT317-PR5, Utrecht, Netherlands) was used to determine the relative number of TNF-aexpressing cells in the spleen lymphocytes. Cells were diluted in RPMI-1640 medium, which contained an appropriate stimulus, such as 2 mg/ml PPD, 2 mg/ml Ag85B, 4 mg/ml Ag85B or 2 mg/ml phytohemagglutinin (PHA). Then 2.5  105 cells were plated to each well of the ELISPOT following the manufacturer’s instructions. The spots were finally counted microscopically. Wells with less than 10 spots were not used for calculations. 4.7. ELISA assay for IFN-g, IL-2 and IL-10 Suspensions obtained from stimulation were used for cytokine ELISA assay. The level of IFN-g, IL-2 and IL-10 were determined by using mouse IFN-g ELISA Kit (Dakewe Biotech Company Limited, Shenzhen, China), mouse IL-2 ELISA Ready-SET-Go Kits (eBioscience, San Diego, CA) and mouse IL-10 ELISA Ready-SET-Go Kits (eBioscience, San Diego, CA), respectively. Experiments were carried out according to the manufacturer’s protocol. 4.8. Flow cytometry analysis for spleen lymphocytes Single-cell suspensions were prepared from the spleens aseptically. 2  106 cells in 2 ml volume were cultured at the presence of stimulus (2 mg/ml PPD or 2 mg/ml Ag85B) at 37  C in a humidified CO2 incubator as described above. After 30 h, cells were harvested and resuspended in the phosphate buffer solution (PBS). Then the cells were incubated with FITC-conjugated anti-mouse CD4 and PEconjugated anti-mouse CD8 mAbs (Biolegend, San Diego, CA) for 30 min at 4  C. The cells were washed twice in PBS and analyzed with a flow cytometer (BD Calibur). 4.9. ELISA assay for Ag85B-specific IgG The sera were collected for antibody detection. ELISA plates (Maxisorb, type 96F; Nunc, Roskilde, Denmark) were coated with Ag85B (1mg/well) overnight at 4  C. Free binding sites were blocked with 300ml/well PBS containing 1% bovin serum albumin (BSA) for 2 h at 37  C. The plates were then washed with PBS containing 0.05% Tween-20 for three times. Serum samples were added at serial twofold dilutions (beginning at a 1/100 dilution) for 1 h at 37  C and washed (as described above), followed by adding 100ml/ well HRP-conjugated goat anti-mouse IgG (Ding Guo Biotechnology, Beijing, China), IgG1 or IgG2b (Jackson ImmunoResearch) diluted at recommended dilutions in PBS, respectively. The plates were incubated for 1 h at 37  C, washed and developed with 0.1M citrate-phosphate buffer, pH 5.0, containing 1 mg/ml o-phenylenediamine and 0.03% hydrogen peroxide. The reactions were stopped by adding 1N H2SO4 (50ml/well) and the OD was read at 450 nm. Antibody titers were expressed as reciprocal end point titers. 4.10. Data analysis Data for each experiment were expressed as a mean (SEM) and the results were analyzed for statistical significance using unpaired Student’s t-tests from the software package SPSS 13.0 (Apache

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