A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice

Available online at www.sciencedirect.com

Immunology Letters 117 (2008) 136–145

A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice Siguo Liu a,∗,1 , Qiang Gong a,b,1 , Chunlai Wang a,f,g , Huifang Liu a , Yong Wang a,c , Sheping Guo a , Weili Wang d , Jiandong Liu a , Meili Shao a,c , Lei Chi a , Kun Zhao a , Zhenguo Wang d , Yuanxiang Shi e , Ying Huang e , Aman guli e , Chunsheng Zhang e , Xiangang Kong a,∗∗ a

Division of Bacterial Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15000, PR China b He Nan University of Science & Technology, Food and Bioengineering. Luo Yang 471003, PR China c Northeast Agricultural University, Harbin 150030, PR China d Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun 130062, PR China e Urumqi Work General Station of Animal Veterinary Quarantine and Grassland, Urumqi 830063, PR China f Animal Husbandry Research Center, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, PR China g Northeast Forestry University, Harbin 150040, PR China Received 10 September 2007; received in revised form 27 December 2007; accepted 15 January 2008 Available online 12 February 2008

Abstract Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB). The proteins Ag85B, MPB64, and ESAT-6 are the major immunogenic antigens of M. bovis; these proteins play important roles in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed various DNA vaccines with the genes encoding the three antigens mentioned above. This procedure involved the following steps: fusion of two genes (pcDNA-MPB64-Ag85B, pcMA), fusion of three genes (pcDNA-MPB64-Ag85BESAT-6, pcMAE), bivalent combinations (pcDNA-Ag85B + pcDNA-MPB64, pcA + M), and trivalent combinations (pcDNA-Ag85B + pcDNAMPB64 + pcDNA-ESAT-6, pcA + M + E). The immune response to the DNA vaccines was evaluated based on serum antibody titers, lymphocyte proliferation assay, and titers of the cytokines interferon-␥ (IFN-␥) and interleukin-2 (IL-2). The protective efficacy following challenge with a virulent M. bovis strain, C68001, was evaluated based on survival rate, bacterial loads in lung tissue, and histopathologic changes. A significant 2-fold increase in serum antibody levels was observed in mice vaccinated with fusion DNA (two or three genes). Furthermore, the lymphocyte proliferation (SI) values and the levels of IFN-␥ and IL-2 were higher in mice vaccinated with fusion DNA (two or three genes) than in those immunized with polyvalent combination DNA vaccines (P < 0.05). Additionally, the fusion DNA vaccines provided protection that was superior to that provided by the polyvalent combination DNA vaccines following challenge with M. bovis strain C68001. The protective efficacy of the fusion DNA vaccines in mice immunized three times was equivalent to the protective efficacy in mice immunized once with the Bacillus Calmette–Guerin (BCG) vaccine. This suggests that fusion DNA vaccine represent a promising approach for the prevention of bTB. © 2008 Elsevier B.V. All rights reserved. Keywords: Mycobacterium bovis; DNA vaccine; Immune efficacy

1. Introduction Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic consumptive zoonosis and is widely distributed in ∗

Corresponding author. Tel.: +86 451 859 35076; fax: +86 451 827 33132. Corresponding author. Tel.: +86 451 859 35000; fax: +86 451 827 33132. E-mail addresses: siguo [email protected] (S. Liu), [email protected] (X. Kong). 1 Both authors contributed equally to this work. ∗∗

0165-2478/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2008.01.008

many countries, particularly in developing countries [1]. It is not only a major cause of devastating economic loss to farming industries but also a serious threat to human health [2]. Approximately 94% of the human population lives in countries where no or limited measures are taken to control bTB in cattle and buffaloes [3]. In developing countries, bTB accounts for more than 10% of TB cases in humans [4]. Thus, the prevention and control of bTB is important to public health. Use of tuberculin purified protein derivative (PPD) for skin test in cattle has become an integral part of the bTB eradication program in devel-

S. Liu et al. / Immunology Letters 117 (2008) 136–145

oped countries. Although, this method effectively controlled the occurrence and prevalence of bTB in some countries, it cannot be applied in other countries or regions [5]. Most developing countries cannot afford the economic burden resulting from a strategy involving cattle slaughter. Moreover, M. bovis, whose natural hosts include wild animals [2], continues to pose a threat to the countries and regions where bTB has been effectively controlled. This leads to the recurrence of bTB in these countries and regions, and presents an enormous difficulty and challenge for bTB prevention. Although, there are reports of the prevention of bTB by using Bacillus Calmette–Guerin (BCG) vaccine, the protective effect achieved through this method is not ideal [6–8]. Thus, research and development of novel vaccines is required to provide an effective means for controlling bTB. DNA vaccines, which can induce systemic immune responses, are currently the subject of intense investigation in the field of vaccine research [9]. Currently, candidates for tuberculosis DNA vaccine antigens include ESAT-6 [10,11], MPB64 [10], antigen Ag85 complex [10,12–14], and hsp65 [15]. Many studies have demonstrated that the low molecular weight secretory protein ESAT-6 is expressed only in the M. tuberculosis complex (which includes M. tuberculosis, M. bovis, M. microti, and M. africanum) and a few other pathogenic mycobacteria, and is not expressed in BCG and most other nonpathogenic mycobacteria [16,17]. The ESAT-6 protein, in which a large quantity of B and T cell epitopes are present, is an strongly antigenic protein [18,19]. A DNA vaccine based on the ESAT-6 gene not only induced the production of high levels of interferon (IFN)-␥ in mice [11] but also reduced the bacterial load in the murine lung after challenge with virulent strains [10,11]. The Ag85 complex is an important secretory protein of M. tuberculosis [20]; it comprises Ag85A, Ag85B, and Ag85C, which are encoded by three different genes. Ag85B, a secretory protein produced in the early stage of M. tuberculosis culture, possesses mycolic acid transferase activity, which is associated with cell wall synthesis. It has the strongest immunogenicity and the highest level of expression among the secretory proteins of M. tuberculosis. A DNA vaccine based on the Ag85B gene could not only elicit a T-helper (Th)-1 response and generate high levels of IFN-␥ in the host but also resisted challenge with a virulent strain of M. tuberculosis [21,22]. MPT64, also called MPB64, is a protein that is secreted during the exuberant cell growth and division stage of M. tuberculosis culture. It contains a major histocompatibility complex (MHC) class I molecular recognition site and the epitope of CD8+ T-cells, and can induce the production of specific cytotoxic T lymphocytes (CTLs) in mice [23–26]. A DNA vaccine based on MPT64 gene was shown not only to elicit high levels of humoral and cellular immune responses but also to effectively reduce the bacterial load in the lung [10]. However, there are large differences between the immune efficacies of monovalent DNA vaccines constructed using these genes [10,27]. Their protective efficacy hardly exceeds that of BCG [28], whereas polyvalent combination and fusion DNA vaccines demonstrate a relatively promising prospect of applica-

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tion as they have an immune efficacy that is obviously superior to that of monovalent DNA vaccines [29–34]. Such vaccines might form the basis of an effective strategy for the prevention of tuberculosis in the future. In this study, we used the secretory antigenic genes ag85b, mpb64, and esat-6 of M. bovis as the basis for construction of the polyvalent combinations (divalent or trivalent) and multigenic fusion (two or three genes) DNA vaccines. We adopted an animal model based on BALB/c mice, which were immunized by intramuscular injection and challenged by inhalation. The results, comprising detection of immunological indicators and measurement of the bacterial load in the lung after challenge, the number of surviving mice, and histopathologic indicators, suggested that multigenic fusion DNA vaccines could improve the immunogenicity and efficacy of immune protection against bTB. This finding has laid a foundation for further research and development of vaccines against M. bovis infection. 2. Materials and methods 2.1. Bacterial strains, sera, and recombinant proteins M. bovis-Vall´ee III strain, C68001 strain, and PPD were purchased from the Chinese Institute of Veterinary Drug Control (IVDC). Rabbit anti-bovine PPD serum was prepared as described previously [35]. The recombinant protein MPB64Ag85B-ESAT-6 (rMAE) was prepared according to a method described previously [36]. This protein was used as a coating antigen for enzyme-linked immunosorbent assay (ELISA) and to stimulate lymphocyte proliferation and differentiation. The concentration of the protein was 0.65 mg/ml. 2.2. Target gene amplification Primers were designed according to the nucleotide sequences of the mpb64, ag85b, and esat-6 genes of the M. bovis strain AF2122/97 (GenBank accession number NC002945) [37]. Table 1 lists the primer sequences. Genomic DNA of the M. bovis-Vall´ee III strain was extracted as described previously [39]. The target gene fragments were amplified using the genomic DNA as a template. The mpb64 gene was amplified using primer pairs 1&2 and 1&3, resulting in fragment I (containing the initiation and termination codons) and fragment II (containing the initiation codon and linker sequences). The ag85b gene was amplified using primer pairs 4&6 and 5&7, resulting in fragment III (containing the initiation and termination codons), fragment IV (containing linker sequences and the termination codon), and fragment V (containing linker sequences). The esat-6 gene was amplified using primer pairs 8&10 and 9&10, resulting in fragment VI (containing the initiation and the termination codons) and fragment VII (containing the termination codon). The amplified products were purified using a gel extraction mini kit (Shanghai Watson Biological Engineering Company, Shanghai, China). The chimeric gene fragment mpb64-ag85b was obtained by splicing

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Table 1 Primer sequences of the mpb64, ag85b and esat-6 gene Names

Oligonucleotides primer sequences

PCR product size (bp)

MPB64 forward (1#) MPB64 reverse (2#) MPB64 reverse (3#) Ag85b forward (4#) Ag85b forward (5#) Ag85b reverse (6#) Ag85b reverse (7#) ESAT-6 forward (8#) ESAT-6 forward (9#) ESAT-6 reverse (10#)

5 -GGCGGCGGTACC|ATG|CGTTATCTGATAGCGAC-3a

684

5 -GCGAATTCTTAGATTGCCAGCGGCGGAATG-3b 5 -GGAACCTGGAGATGGGACCAATACCTG-3c 5 -GCGCGGTACC|ATG|ACAGACGTGAGCCGAAAG-3d 5 -TCTCCAGGTTCCACAGACGTGAGCCGAAAG-3e 5 -GTGGGGAATTCCTAGCCGGCGCCTAACGAAC-3f 5 -GAGGGATCCTGGAGAGCCGGCGCCTAAC-3g 5 -ACGGGATCC|ATG|CAGCAGTGGAATTCC-3h 5 -ACGGGATCCGAGCAGCAGTGGAATTTC-3i 5 -GCCGAATTCCTATGCGAACATCCCAGTG-3J

986

288

Note: Letters a, d: the KpnI recognition sequence is underlined; letters b, f, j: the EcoRI recognition sequence is underlined; letters g, h, I: the BamHI recognition sequence is underlined; letters c: a linker gene encoding four amino acid residues (-SPGS-) was introduced into the fusion genes. Due to the corner effect of proline and the flexibility effect of glycine, an ␣-helix cannot form and thus the expressed proteins can maintain their spatial structure and relatively independent biological functions [38]. The linker is in bold. Letter e: the reverse complementary sequence of the linker is in bold; Letters a, d, h: the start codon is boxed; letters b, f, j: the stop codon is italic.

with overlap extension using polymerase chain reaction (PCR). The purified fragments II and IV were used as templates and primers 1 and 6 were used; the resulting fragment was termed VIII (containing the initiation and termination codons). The chimeric gene fragment mpb64-ag85b was obtained using the purified fragments II and V as templates and primers 1 and 7; the resulting fragment was termed IX (containing an initiation codon).

isothiocyanate (FITC)-labelled goat anti-rabbit IgG (1:100 dilution, Sigma, St. Louis, MO, USA) containing 1% Evans blue was added; the samples were incubated in a moist chamber for 1 h at 37 ◦ C, and were then observed in a drop of basic glycerine under an inverted microscope to visualize blue-green fluorescence in Sp2/0 cells.

2.3. Construction and in vitro expression of recombinant eukaryotic plasmids

Female BALB/c mice (n = 112), 6–8 weeks old and weighing 18–20 g, were obtained from the second affiliated hospital, Harbin Medical University. The animals were randomly assigned to immunization groups containing 16 mice each. The mice were immunized with 100 ␮g of the DNA vaccine for each immunization group, the mice were injected into both tibialis anterior muscles. The mice in the bivalent combination group (pcDNA-Ag85B + pcDNA-MPB64, pcA + M) received 50 ␮g of each vaccine construct and those in the trivalent combination group (pcDNA-Ag85B + pcDNAMPB64 + pcDNA-ESAT-6, pcA + M + E) received 33 ␮g of each vaccine construct. Mice in the negative control groups were given PBS (0.01 M, pH 7.2) or empty vector pcDNA3.1(+). The animals in each of the above groups were immunized three times at 2-week intervals. In the positive control group, mice were inoculated subcutaneously with 1 × 106 colony-forming units (CFUs) of BCG at the time of initial vaccination [42].

The purified gene fragments I, III, VI, and VIII were ligated into pcDNA3.1(+) (Invitrogen, San Diego, CA, USA), resulting in recombinant plasmids pcDNA-MPB64 (pcM), pcDNA-Ag85B (pcA), pcDNA-ESAT-6 (pcE), and pcDNAMPB64-Ag85B (pcMA), respectively. Gene fragment IX was ligated to pcDNA3.1(+), resulting in the recombinant plasmid pcDNA-MPB64/Ag85B. The gene fragment VII was subcloned downstream of the ag85b gene in pcDNA-MPB64/Ag85B, resulting in pcDNA-MPB64-Ag85B-ESAT-6 (pcMAE). The recombinant plasmids pcM, pcA, pcE, pcMA, and pcMAE were confirmed by sequencing. The plasmids were prepared on a large scale using the alkaline lysis method, and the plasmid preparations were adjusted to 1 ␮g/␮l using phosphate-buffered saline (PBS: 0.01 M, pH 7.2) for further experiments [40]. One day before transfection, mouse myeloma Sp2/0 cells (purchased from The Institute of Cell Biology, Chinese Academy of Sciences) at logarithmic growth phase were seeded onto 24-well plates (Greiner Bio-One, Longwood, Germany) and grown at 37 ◦ C, in 5% CO2 until 70–80% confluence. They were then transfected with 0.8 ␮g of pcM, pcA, pcE, pcMA, pcMAE, and empty vector pcDNA3.1(+) using 2 ␮l of Lipofectamine 2000 (Gibco, Grand Island, NJ, USA) followed by indirect immunofluorescent testing after 72 h [41]. Briefly, the cells were fixed with methanol/acetone for 15 min at room temperature, followed by the addition of 200 ␮l rabbit anti-PPD polyclonal antibody (1:40 dilution), and incubated in a moist chamber for 1 h at 37 ◦ C. Subsequently, 200 ␮l of fluorescein

2.4. Immunization of mice

2.5. Serum antibody levels After immunization, blood samples were drawn from the mice and serum was obtained. The serum antibody titers were determined using indirect ELISA [43]. Briefly, ELISA microtiter plates (eBioscience, San Diego, CA, USA) were coated with 10 ␮g/ml rMAE protein, followed by blocking the nonspecific binding with 100 ␮l of 1% gelatin (Sigma, St. Louis, USA) for 2 h. This was followed by the addition of 50 ␮l of serum (1:100 dilution), and the samples were incubated in a moist chamber at 37 ◦ C for 1.5 h. The plates were washed three times with PBST (0.01 M PBS-0.05% Tween-80, pH 7.2), and goat anti-mouse

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IgG-horseradish peroxidase (HRP) (Sigma–Aldrich, St. Louis, MO, USA) was added. The plates were then incubated at 37 ◦ C for 1.5 h. Plates were washed three times with PBST, then 50 ␮l ortho-phenylene diamine (OPD, Sigma) was added and incubated for 10 min; enzyme activity was stopped by adding an equal volume of 2 M H2 SO4 , and the absorption was measured at 492 nm. Antibody titers were determined for up to 7 weeks before challenge. For IgG isotype detection, serum specimens obtained in the 4th and 7th weeks were assayed by ELISA with HRP-conjugated goat anti-mouse IgG1 and IgG2a. 2.6. Splenic lymphocyte proliferation assay Two weeks after each immunization, the spleens from the 2 mice in each group were harvested under aseptic conditions and splenic lymphocyte suspensions were prepared. The cell concentrations were adjusted to 1 × 107 cells/ml. An aliquot (50 ␮l) of cell suspension was seeded in a 96-well plate (Greiner Bio-One, Longwood, Germany). Each experiment was repeated three times. Each well received 50 ␮l of 10 ␮g/ml rMAE protein (experimental well) or 50 ␮l of RPMI 1640 medium (Gibco, Grand Island, USA) (negative control), and the plates were incubated at 37 ◦ C in 5% CO2 . After 36 h, 10 ␮l of 5 mg/ml 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT; Sigma–Aldrich) was added to each culture well and incubated for 3 h. After centrifugation for 10 min, the supernatant was discarded and 150 ␮l of dimethyl sulfoxide (DMSO) was added to the pellet and incubated for 10 min until the crystals dissolved. The optical density (OD) value of each well was measured using a microplate reader (ELX80; BioTek Instruments, Bioian model 680) with a test wavelength of 570 nm. The stimulation index (SI) was estimated using the following equation: SI = OD experimental well/OD negative control well [10]. 2.7. IFN-γ and interleukin (IL)-2 assays Two weeks after each immunization, splenic lymphocytes activated by the rMAE protein were prepared as described above. The cells were incubated at 37 ◦ C in 5% CO2 for 72 h; the supernatants were then harvested and stored at −20 ◦ C. The levels of IFN-␥ and IL-2 in the supernatant were detected using a commercial ELISA kit (Jing Mei Biotech Company, Beijing, China) according to manufacturer’s instructions [44].

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Lowenstein–Jensen medium [42], and the plates were incubated at 37 ◦ C for 4–6 weeks; the number of CFU was then counted [30,47]. 2.9. Histopathology and survival experiment Lung samples were subjected to hematoxylin-eosin (HE) staining for the observation of histologic changes. Briefly, following challenge, the right lung of each mouse was excised, fixed in 10% formalin, embedded in paraffin, and cut into 5–6 ␮m sections. All sections were heated at 56 ◦ C for 25 min, deparaffinized in xylene, rehydrated with graded alcohols, and then stained with HE for histological observation using light microscopy (Olympus). The capacity of the DNA vaccines to elicit a sustained protective immune response was also assessed in long-term survival studies. For this experiment, the post-challenge survival periods of the mice immunized with various DNA vaccines were compared with those of the BCG-vaccinated and negative controls. 2.10. Statistical analysis The data from the experiments were expressed as mean ± standard deviation (S.D.). Statistical analysis was conducted with SAS software (Version 9.0; SAS Inst., Cary, NC, USA). Analysis of variance (ANOVA) was used to determine the significance of differences in means between the multiple experimental groups. Differences with P < 0.05 were considered significant. 3. Results 3.1. In vitro expression of recombinant plasmids Sp2/0 cells transfected with recombinant plasmids showed homogeneous blue–green fluorescence under an inverting microscope, demonstrating that the M. bovis genes, ag85b, mpb64, esat-6, mpb64-ag85b, and mpb64-ag85b-esat-6 were transfected in vitro into Sp2/0 cells and that the cells expressed the target proteins. The target proteins were observed to bind specifically to the rabbit anti-bovine PPD polyclonal antibodies. In contrast, the Sp2/0 cells transfected with pcDNA3.1(+) did not fluoresce (Fig. 1). 3.2. Detection of serum antibodies

2.8. Challenge and bacterial load in lung tissue There are several different challenge models available, such as aerogenical, tracheal, intravenous, and intraperitoneal. In this study, the mice were challenged 3 weeks after the 3rd DNA immunization with the virulent strain C68001 of M. bovis (approximately 1 × 103 CFU) in an aerosol-generating chamber, as described previously [45,46]. Immunized and control animals were sacrificed on day 21 post-challenge. The left lungs extracted from the mice were homogenized in 0.05% PBST, and serially diluted 10-fold. Following this, 100 ␮l of the diluted samples (10–3 , 10–4 , and 10–5 ) were plated on modified

The levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of the immunized mice indicated that strong humoral responses had been generated by the DNA vaccine constructs. After immunization, the serum antibody levels in the pcA+M-, pcA+M+E-, pcMA-, and pcMAE-vaccinated groups were higher than those in the BCG-vaccinated group; the antibody levels in the pcMA- and pcMAE-vaccinated groups were significantly higher than those in the pcA+M-, pcA+M+E-, and BCG-vaccinated groups (P < 0.05) (Fig. 2). In addition, the antibody isotype results clearly suggested that at the 4th and 7th week after immunization, in the mice vaccinated with the poly-

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Fig. 1. Immunofluorescence in Sp2/0 cells transfected with recombinant plasmids. a: Cells transfected with pcA; b: pcM; c: pcE; d: pcMA; e: pcMAE. Cells transfected with recombinant plasmids showed homogeneous blue-green fluorescence; f: Cells transfected with vector pcDNA3.1(+) did not fluoresce.

valent combination, multigenic fusion, and BCG vaccines, the levels of IgG2a were higher than the levels of IgG1. The levels of IgG2a in the pcMA- and pcMAE-vaccinated groups were significantly higher than those in the pcA+M-, pcA+M+E-, and BCG-vaccinated groups (P < 0.05) and in the negative control groups (P < 0.01). The levels of IgG1 and IgG2a were higher at the 7th week than at the 4th week (Fig. 3).

pcA + M + E-, pcMA-, pcMAE-vaccinated groups were similar to those of the BCG-vaccinated group (P > 0.05). After the second and third immunizations, however, the SI value in the pcMA- and pcMAE-vaccinated groups was significantly higher than that in the pcA + M-, pcA + M + E-, and BCG-vaccinated groups (P < 0.05) and the negative control groups (P < 0.01), with no difference in the values between the pcMA- and pcMAE-vaccinated groups (P > 0.05).

3.3. Splenic lymphocyte proliferation assay 3.4. IFN-γ and IL-2 secretion The cell-mediated immune responses of the mice were assessed at three time points after vaccination during each experiment, and the results obtained are shown in Fig. 4. In each experiment, the SI values for the polyvalent combination, multigenic fusion, and BCG-vaccinated groups were consistently higher than those for the negative control groups. After the first immunization, the SI values of the pcA + M-,

Fig. 2. Dynamic changes of specific serum antibodies in immunized mice Following the first immunization, serum antibody levels were measured by indirect ELISA weekly until 7 weeks. Mice were immunized with pcA + M vaccine( ); pcA + E + M vaccine ( ); pcMA vaccine ( ); pcMAE vaccine ( ); BCG ( ); pcDNA3.1(+) vector ( ) ). and PBS (

After rMAE protein stimulation, the levels of IFN-␥ and IL2 secreted by the mouse splenic lymphocytes were increased

Fig. 3. Effect of DNA vaccination on IgG1 and IgG2a levels in mice immunized with various vaccines as described in the Materials and Methods. IgG1 and IgG2a levels at the 4th week (A) and 7th week (B) were determined by ELISA. *P < 0.05, **P < 0.01.  P < 0.05.

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Fig. 4. MTT detection of splenic lymphocyte proliferation in immunized mice The rMAE protein was administered to stimulate mouse splenic lymphocytes 2 weeks after each immunization. FI: first immunization; SI: second immunization; TI: third immunization. *P < 0.05, **P < 0.01.

in the mice vaccinated with the polyvalent combination, multigenic fusion, and BCG vaccines. After the first immunization, no significant differences were detected among these groups (P > 0.05). After the second and third immunization, the levels of IFN-␥ and IL-2 were similar in the pcMA- and pcMAEvaccinated groups, but were significantly higher in the pcA + M-, pcA + M + E-, and BCG-vaccinated groups (P < 0.05) and the negative control groups (P < 0.01). After the third immunization, the IFN-␥ and IL-2 levels in the pcA + M-vaccinated group were slightly lower than those in the BCG-vaccinated group, and were slightly higher in the pcA + M + E-vaccinated group than in the BCG-vaccinated group (Fig. 5 A and B). 3.5. Bacterial loads in lung tissue

Fig. 5. ELISA detection of mouse splenic lymphocyte-secreted IFN-␥ (A) and IL-2 (B) levels The rMAE protein was administered to stimulate mouse splenic lymphocytes 2 weeks after each immunization. The results are expressed as mean ± S.D. FI: first immunization; SI: second immunization; TI: third immunization. *P < 0.05, **P < 0.01.

vaccinated group showed congestion, widening of alveolar septa, marked telangiectasia, and pink alveolar fibrin exudates.

The log10 CFUs recovered from the lungs in the pcA + M, pcA + M + E-, pcMA-, pcMAE-, and BCG-vaccinated groups were significantly lower than those in the negative control groups (P < 0.05). The log10 CFUs observed in the lungs of animals immunized with pcMA (2.83 ± 0.03) and pcMAE (2.87 ± 0.04) were comparable to those in the lungs of animals immunized with BCG (2.81 ± 0.04). However, the log10 CFUs in the lungs of animals immunized with the multigenic fusion vaccines were lower than those in the pcA + M- (3.23 ± 0.03) and pcA + M + Evaccinated (3.18 ± 0.03) groups (P < 0.05) (Table 2).

3.7. Survival studies

3.6. Lung tissue pathology

Groups

Lung CFUs (log10 )a

pcA + M pcA + M + E pcMA pcMAE BCG PBS EV

3.23 3.18 2.83 2.87 2.81 4.33

In the negative control groups, the HE-stained lung tissue sections revealed (Fig. 6) severe pathologic alterations after challenge with the virulent M. bovis strain C68001. These alterations included substantial alveolar fibrin exudation and fibrous pneumonia. The pcMA-, pcMAE-, and BCG-vaccinated groups exhibited relatively mild pathologic alterations, i.e., telangiectasia and mild fibrin exudation. Pathological changes were more severe in the pcA + M- and pcA + M + E-vaccinated groups than in the pcMA- and pcMAE-vaccinated groups. The pcA + Mvaccinated group presented with telangiectasia and infiltration of a small number of lymphoid cells, and the pcA + M + E-

The results of the survival experiment showed that the survival period of mice vaccinated with the polyvalent combination, multigenic fusion, and BCG vaccine was significantly longer than that of mice in the negative control groups. Vaccination with the two fusion DNA vaccines (pcMA and pcMAE) significantly extended the survival period compared with that polyvalent comTable 2 Protective immune responses to DNA vaccines (mean log10 CFU/mg ± S.D.)

± ± ± ± ± ±

0.03 0.03 0.03 0.04 0.04 0.03 4.37 ± 0.03

Lung protectionb 1.10* 1.15* 1.54* 1.50* 1.52* −0.04 –

a Bacterial counts in the lungs (or spleens) of mice immunized with the indicated vaccines and then aerogenically challenged 3 weeks after the final vaccination. The mice were sacrificed for CFU determination 3 weeks after the challenge. Data are represented as the mean CFU ± standard error. b The reduction in bacterial counts, relative to nonimmunized mice, in the lungs of vaccinated mice. Asterisks represent statistically significant (P < 0.05).

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Fig. 6. Histopathology of lung from mice in various groups after challenge with M. bovis C68001 strain (HE staining; 200× magnification) 5a: pcM + A DNA vaccine; 5b: pcM + A + E DNA; 5c: pcMA fusion vaccine; 5d: pcMAE fusion vaccine; 5e: BCG; 5f: pcDNA3.1(+); 5g: PBS; 5h: control.

Fig. 7. The survival of mice immunized with fusion and polyvalent DNA vaccines, BCG, pCDNA3.1(+) and PBS following challenge with M. bovis C68001 strain. The mice were observed for mortality for 300 days. Ten mice per group were used in this analysis. **P < 0.01.

bination DNA vaccines (pcA + M and pcA + M + E) (P < 0.01). All mice died before 200 days post-challenge in the negative control groups. By the 300th day post-challenge, five mice (50%) had survived in the pcMA- and BCG-vaccinated groups and four mice (40%), in the pcMAE-vaccinated group. Only one mouse (10%) survived in the DNA-A + M-vaccinated group by the 300th day. The results suggest that when administered three times, the two gene fusion DNA vaccines can significantly prolong the survival period than the polyvalent DNA vaccines. Furthermore, their efficacy is equivalent to that observed when BCG is administered once (Fig. 7). 4. Discussion M. bovis, an intracellular bacterium, is mainly associated with induction of a cellular immune response [48,49]. Protective immunity against mycobacterial infection is mediated by interactions between specifically primed CD4 + and CD8 + T cells and activated macrophage effector cells harboring the intracellular pathogen [50,51]. Mycobacterial infection can induce the

generation of numerous cytokines, particularly Th1 cytokines such as IFN-␥ and IL-2, which activate the macrophages. The activated macrophages bind CD4 + and CD8 + T cells and promote the formation of tuberculous granuloma, thus inhibiting the spread of M. tuberculosis. IFN-␥ is a critical cytokine in the control of M. tuberculosis infection. The level of IFN-␥ generated is an important indicator of the protective efficacy of DNA vaccines against M. bovis [52–54]. The production of IFN-␥ is induced by DNA vaccination against M. bovis, and is positively correlated with protective efficacy [55]. Previous studies have shown that DNA vaccine constructed using M. bovis genes such as those encoding Ag85B, MPB64, ESAT-6, and MPB83 induced the production of high levels of IFN-␥, and produced relatively good protective efficacy in a small animal model [21,56,57]. However, the degree of protective efficacy did not reach that achieved using BCG [21,57–60]. Luo et al. [61] constructed an Ag85B-MPT64 fusion gene vaccine to immunize mice, and found that it could elicit PPD-specific IgG and a lymphocyte proliferation immune response, and produced relatively high levels of IFN-␥. Moreover, the IFN-␥ level in the pcDNA/AM group was notably higher than that in the pcDNA/Ag85B and pcDNA/MPT64 groups due to improved immunogenicity resulting from the fusion of genes encoding Ag85B and MPT64. In order to enhance the protective efficacy of DNA vaccines, researchers have explored numerous strategies, including the use of polyvalent DNA vaccine, multigenic fusion DNA vaccines, and primary immunization with polyvalent DNA with booster immunization with BCG. The protective efficacy of polyvalent M. bovis DNA vaccines and two gene fusion DNA vaccines is significantly superior to that of monovalent vaccines, and the levels of IFN-␥ when the former are used are higher than those observed with BCG. In addition, the level of protection achieved using polyvalent and fusion vaccines is equivalent to that achieved using BCG after challenge [10,29,30,34,62]. The

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protective efficacy resulting from primary vaccination with a polyvalent DNA vaccine boosted with BCG exceeded that of BCG alone [63]. In this study, both polyvalent combined DNA vaccines (pcA + M and pcA + M + E) and multigenic fusion DNA vaccines (pcMA and pcMAE) induced the production of high levels of IFN-␥ in splenic cells from immunized mice; these levels were significantly higher than those observed in the control groups (P < 0.01). Moreover, the IFN-␥ level observed for the multigenic fusion DNA vaccines exceeded that observed for BCG (P < 0.05). The level of IFN-␥ secreted by splenic cells from the mice immunized with multigenic fusion DNA vaccines reached 1100 pg/ml, which exceeded that observed for M. tuberculosis quadrivalent DNA vaccines (encoding the Ag85B, MPT64, MPT70, and TB10.4 antigens) reported by Li et al. [64]. However, this was equivalent to the level of 1250 pg/ml that was obtained using quadrivalent DNA vaccines for primary immunization and BCG for booster immunization. Zhu et al. [60] constructed pcD85B, pcDMPT64, and pcD85B + pcDMPT64 DNA vaccines to immunize C57BL/6 mice and challenged them with M. tuberculosis. The IFN-␥ level observed for pcD85B, pcDMPT64, and pcD85B + pcDMPT64 DNA vaccines was higher than that observed for the multigenic fusion DNA vaccines constructed in the current study. However, the lung protection value after challenge was only 1.2, whereas the value achieved using the multigenic fusion DNA vaccine pcMA was 1.54 and that achieved using pcMAE was 1.50. A logarithmic difference in the value of the bacterial load in the lung of higher than 0.7 is considered to represent protective efficacy [65]. The levels of different subclasses of antigen-specific IgG antibodies, and particularly the IgG1/IgG2a ratio, partly reflects the T helper lymphocyte response. An IgG2a/IgG1 > 1 or IgG2a/IgG1 < 1 indicates a Th1 or Th2 response, respectively. The cytokines produced by a Th1 cellular response, such as INF-␥, can enhance the expression of IgG2 [66], while those produced by a Th2 cellular response, such as IL-4, can induce the expression of IgG1, IgG3, and IgG4 [67]. In this study, we detected the subclasses of IgG, and found that the level of IgG2a (Th1) was higher than that of IgG1 (Th2) in both the DNA vaccine-immunized groups (pcA + M, pcA + M + E, pcMA, and pcMAE) and the BCG-immunized group, showing that IgG2a levels were predominant in the BALB/c mice. This finding is consistent with that reported by Ulmer et al. [13], indicating that the immune response induced by DNA vaccines is based chiefly on a Th1 type cellular immune response. The SI value and the IFN-␥, IL-2, and specific IgG antibody levels were slightly higher in the mice immnunized with a vaccine containing two fused genes than in those immunized with a vaccine containing three fused genes. This may be because a BamH I site replaced a linker sequence between the ag85b and esat-6 genes in pcMAE, thus the expression of the protein by pcMAE may have resulted in mutual interference between the three domains (MPB64, Ag85B, and ESAT-6); in particular some T cell epitopes may have been covered. Therefore, the ability of fusion DNA vaccines to induce immune responses does not depend only on the number of genes; it may also depend on

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the interactions between the genes. This is similar to the “antigen competition” that often occurs in combination DNA vaccination [68]. However, in this study, we did not observe “antigen competition” phenomena when using the polyvalent combined DNA vaccines (pcA + M and pcA + M + E). Although the protective efficacy and IFN-␥ level were lower in the polyvalent DNA vaccine groups than in the multigenic fusion DNA vaccine groups, they were higher than those obtained by vaccination with three single DNA vaccines (pcA, pcE, or pcM, data not shown). Morris et al. [69] also showed that no “antigen competition” occurred when a polyvalent combination DNA vaccine (containing ESAT6, MPT-64, MPT-63, and KatG constructs) was used based on the detection of the immune response to each component of the mixture. As mentioned in the Introduction, our main goal in this study was to develop an effective vaccine for bTB. The multigenic fusion DNA vaccines and polyvalent DNA vaccines we constructed performed well in terms of serum antibody titers, lymphocyte proliferation assay results, IFN-␥ and IL-2 levels and protective efficacy in comparison with BCG. Among the DNA vaccines in this study, the two gene fusion DNA vaccine (pcMA) showed most promise, and provides a valuable reference for the design of future DNA vaccines for M. bovis. Acknowledgements This work was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (No. 2006 CB504400). References [1] Pollock JM, Neill SD. Mycobacterium bovis infection and tuberculosis in cattle. Vet J 2002;163:115–27. [2] Buddle BM, Wedlock DN, Denis M. Progress in development of tuberculosis vaccines for cattle and wildlife. Vet Microbiol 2006;112:191–200. [3] Cousins DV. Mycobacterium bovis infection and control in domestic livestock. Rev Sci Tech Oie 2001;20:71–85. [4] Cosivi O, Grange JM, Daborn CJ, Raviglione MC, Fujikura T, Cousins D, et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4:59–70. [5] Vordermeier HM, Chambers MA, Buddle BM, Pollock JM, Hewinson RG. Progress in the development of vaccines and diagnostic reagents to control tuberculosis in cattle. J Vet 2006;17:229–44. [6] Skinner MA, Wedlock DN, Buddle BM. Vaccination of animals against Mycobacterium bovis. Rev Sci Tech Oie 2001;20:112–32. [7] Wedlock DN, Vesosky B, Skinner MA, Lisle GW, Orime IM, Buddle BM. Vaccination of with Mycobacterium bovis culture filtrate proteins and interleukin-2 for protection cattle against bovis tuberculosis. Infect Immun 2000;68:5809–15. [8] Wedlock DN, Keen DL, Mccarthy AR, Andersen P, Buddle BM. Effect of adjuvants on immune responses of cattle vaccinated with culture filtrate proteins from Mycobacterium tuberculosis. Vet Immunol Immunop 2002;86:79–88. [9] Bonato VLD, Lima VMF, Tascon RE, Lowrie DB, Silva C. Identification and characterization of protective T cells in hsp65 DNA-vaccinated and Mycobacterium tuberculosis infected mice. Infect Immun 1998;66:169– 75. [10] Kamath AT, Feng CG, Macdonald M, Briscoe H, Britton WJ. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun 1999;67:1702–7.

144

S. Liu et al. / Immunology Letters 117 (2008) 136–145

[11] Brandt L, Elhay M, Rosenkrands I, Lindblad EB, Andersen P. ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect Immun 2000;68:791–5. [12] Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Deck RR, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996;2:893–8. [13] Ulmer JB, Liu MA, Montgomery DL, Yawman AM, Deck RR, DeWitt CM, et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination. Vaccine 1997;15:792–4. [14] Baldwin SL, Souza CD, Roberts AD, Kelly BP, Frank AA, Lui MA, et al. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun 1998;66:2951–9. [15] Lowrie DB, Tascon RE, Bonato VL, Lima VM, Faccioli LH, Stavropoulos E, et al. Therapy of tuberculosis in mice by DNA vaccination. Nature 1999;400:269–71. [16] Sorensen AL, Nagai S, Houen G, Andersen P, Andersen AB. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun 1995;63:1710–7. [17] Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis -BCG. Infect Immun 1996;64:16–22. [18] Harboe M, Malin AS, Dockrell HS, Wiker HG, Ulvund G, Holm A, et al. B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis. Infect Immun 1998;66:717–23. [19] Brandt L, Oettinger T, Holm A, Andersen AB, Andersen P. Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protectivie immunity to Mycobacterium tuberculosis. J Immunol 1996;157:3527– 33. [20] Sacchettini JC, Ronning DR. The mycobacterial antigens 85 complex-from structure to function and beyond: response. Trends microbiol 2000;8:441. [21] Lozes E, Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 1997;15:830–3. [22] Kamath AT, Groat NL, Bean AG. Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN gamma) secreting lymphocytes. Clin Exp Immunol 2000;120:476–82. [23] Haslov K, Andersen A, Nagai S, Gottschau A, Sorensen T, Andersen P. Guinea pig cellular immune responses to proteins secreted by Mycobacterium tuberculosis. Infect Immun 1995;63:804–10. [24] Roche PW, Winter N, Triccas JA, Feng CG, Britton WJ. Expression of Mycobacterium tuberculosis MPT64 in recombinant Mycosmegmatis: purification, immunogenicity and application to skin test s for tuberculosis. Clin Exp Immunol 1996;103:226–32. [25] Oettinger T, Holm A, Haslov K. Characterization of the delayed type hypersensitivity inducing epitope of MPT64 from Mycobacterium tuberculosis. Scand J Immunol 1997;45:499–503. [26] Wang Z, Potter BM, Gray AM, Sacksteder KA, Geisbrecht BV, Laity JH. The solution structure of antigen MPT64 from Mycobacterium tuberculosis defines a new family of beta-grasp proteins. J Mol Biol 2007;366:375–81. [27] Natio M, Matsuoka M, Ohara N, Nomaguch H, Yamada T. The antigen 85 complex vaccine against experimental Mycobacterium leprae infection in mice. Vaccine 1999;18:795–8. [28] Orme IM. Current progress in tuberculosis vaccine development. Vaccine 2005;23:2105–8. [29] Agger EM, Rosenkrands I, Olsen AW, Hatch G, Williams A, Kritsch C, et al. Protective immunity to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant system IC31. Vaccine 2006;24:5452–60. [30] Cai H, Tian X, Hu XD, Li SX, Yu DH, Zhu YX. Combined DNA vaccines formulated either in DDA or in saline protect cattle from Mycobacterium bovis infection. Vaccine 2005;23:3887–95. [31] Casellia E, Bonia M, Di LD. A combined bovine herpesvirus 1 gB-gD DNA vaccine induces immune response in mice. J Infect Dis 2005;28:155– 66. [32] Derrick SC, Yang AL, Morri SL. A polyvalent DNA vaccine expressing an ESAT-6-Ag85B fusion protein protects mice against a primary infec-

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

[43]

[44]

[45]

[46]

[47] [48] [49]

[50] [51] [52] [53]

tion with Mycobacterium tuberculosis and boosts BCG-induce protective immunity. Vaccine 2004;23:780–8. Feng CG, Palendira U, Demangel C, Spratt JM, Malin AS, Britton WJ. Priming by DNA immunization augments protective efficacy of Mycobacterium bovis bacillus Calmette–Guerin against tuberculosis. Infect Immun 2001;69:4174–6. Morris S, Kelley C, Howard A, Li ZM, Collins F. The immunogenicity of [0 0 0] single and combination DNA vaccines against tuberculosis. Vaccine 2000;18:2155–63. Metzler B, Mayr M, Dietrich H, Singh M, Wiebe E, Xu QB, et al. Inhibition of arteriosclerosis by T-Cell depletion in normocholesterolemic rabbits immunized with heat shock protein 65. Arterioscler Throm Vasc Biol 1999;19:1905–11. Liu SG, Guo SP, Wang CL, Shao ML, Zhang XH, Guo Y, et al. A novel fusionprotein based indirect enzyme-linked immunosorbent assay for detection of bovine tuberculosis. Tuberculosis 2007;87:212–7. Garnier T, Eiglmeuer K, Camus JC, Medina N, Mansoor H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci USA 2003;100:7877–82. Liu SG, Yu XL, Wang CL, Wu JM, Kong XG, Tu CC. Quadruple antigenic epitope peptide producing immune protection against classical swine fever virus. Vaccine 2006;24:7175–80. Portillo PD, Murillo LA, Patarroyo ME. Amplification of a species-specific DNA fragment Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol 1991;29:2163–8. Sambrook J, Russell D. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbour: Cold Spring Harbour Laboratory Press; 2002. Helou J, Allbritton J, Anhalt GJ. Accuracy of indirect immunofluorescence testing in the diagnosis of paraneoplastic pemphigus. J Am Acad Dermatol 1995;32:441–7. Hernandez YL, Corona DY, Rodriguez SS, Bourzac JFI, Sarmiento ME, Arzuaga NO, et al. Immunization of mice with Mycobacterium tuberculosis genomic expression library results in lower bacterial load in lungs after challenge with BCG. Tuberculosis 2006;86:247–54. Tollefsen S, Vordermeier M, Olsen I, Storset AK, Reitan LJ, Clifford D, et al. DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants. Scand J Immunol 2003;57:229– 38. Denis O, Tanghe A, Palfliet K, Jurion F, Berg TP, Vanonckelen A, et al. Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates a CD4 + and CD8 + T-cell epitope repertoire broader than that stimulated by Mycobacterium tuberculosis H37Rv infection. Infect Immun 1998;66:1527–33. Aldwell FE, Keen DL, Parlane NA, Skinner MA, de Lisle GW. Buddle BM oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums. Vaccine 2003;22:70–6. Derrick SC, Yang AL, Morris SL. A polyvalent DNA vaccine expressing an ESAT6-Ag85B fusion protein protects mice against a primary infection with Mycobacterium tuberculosis and boosts BCG-induced protective immunity. Vaccine 2004;23:780–8. Van-Crevel R, Ottenhoff THM, Vander-Meer JWM. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002;15:294–309. Dietrich G, Viret JF, Hess J. Mycobacterium bovis BCG-based vaccines against tuberculosis: novel developments. Vaccine 2003;21:667–70. Tanghe A, Dsouza S, Rosseels V, Denis O, Ottenhoff THM, Dalemans W, et al. Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect Immun 2001;69:3041–7. Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001;19:93–129. Lowrie DB. DNA vaccines for therapy of tuberculosis: where are we now? Vaccine 2006 a species-specific. Vaccine 2006;24:1983–9. Agger EM, Andersen P. Tuberculosis subunit vaccine development: on the role of interferon-␥. Vaccine 2001;19:2298–302. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon-␥ gene disrupted mice. J Exp Med 1993;178:2243–7.

S. Liu et al. / Immunology Letters 117 (2008) 136–145 [54] Florido M, Goncalves A, Silva RA, Ehlers S, Cooper AM, Appelberg R. Resistance of virulent Mycobacterium avium to gamma interferon-mediated activity suggests additional signals for induction of mycobacteriostasis. Infect Immun 1999;67:3610–8. [55] Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, Hewinson RG. Correlation of ESAT-6 specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infect Immun 2002;70:3026–32. [56] Chamber MA, Stagg D, Gavier WD, Lowrie D, Newell RG, Hewinson RGA. DNA vaccine encoding MPB83 from Mycobacterium bovis reduces M. bovis dissemination to the kidney pf mice and is expressed in primary cell cultures of the European badger. Res Vet Sci 2001;71:119–26. [57] Khera A, Singh R, Shakila H, Raoa V, Dhar N, Narayanan PR, et al. Elicitation of efficient, protective immune responses by using DNA vaccines against tuberculosis. Vaccine 2005;23:5655–65. [58] Mollenkopf HJ, Triebkorn DG, Andersen P, Hess J, Kaufmann SHE. Protective efficacy against tuberculosis of ESAT-6 secreted by a live Salmonella typhimurium vaccine carrier strain and expressed by naked DNA. Vaccine 2001;19:4028–35. [59] Wang QM, Sun SH, Hu ZL, Yin M, Xiao CJ, Zhang JC. Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting. Vaccine 2004;22:3622–7. [60] Zhu DY, Jiang SH, Luo XD. Therapeutic effects of Ag85B and MPT64 DNA vaccines in a murine model of Mycobacterium tuberculosis infection. Vaccine 2005;23:4619–24. [61] Luo XD, Zhu DY, Chen Q, Jiang Y, Jiang S. Immunogenicity of DNA vaccine enco- ding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64. Chin J Cell Mol Immunol 2003;19:592–4.

145

[62] Mauea AC, Waters WR, Palmer MV, Nonnecke BJ, Minion FC, Browne WC, et al. An ESAT-6: CFP10 DNA vaccine administered in conjunction with Mycobacterium bovis BCG confers protection to cattle challenged with virulent M. bovis. Vaccine 2007;25:4735–46. [63] Skinner MA, Buddle BM, Wedlock DN, Keen D, Lisle GW, Tascon RE, et al. A DNA prime-BCG boost vaccination strategy in cattle induces protection against bovis tuberculosis. Infect Immun 2003;71: 4901–7. [64] Li M, Yu DH, Cai H. DNA prime-BCG boost vaccination strategy improved the protective efficacy against M. tuberculosis H37Rv in mice. Prog Biochem Biophys 2007;34:746–53. [65] Orme I, Mcmurray D, Belisle JT. Tuberculosis vaccine development: recent progress. Trends Microbiol 2001;9:115–8. [66] Fujieda S, Zhang K, Saxon A. I-24 plus CD40 monoclonal antibody induces human B cells gamma subclass-specific isotype switch: switching to gamma1, gamma 3, and gamma 4, but not gamma 2. J Immunol 1995;155:2318–28. [67] Kawano Y, Noma T, Yata J. Regulation of human IgG subclass production by cytokines, IFN-gamma and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2. J Immunol 1994;153:4948–58. [68] Delogu G, Li A, Repique C, Collins F, Morris SL. DNA vaccine combinations expressing either tissue plasminogen activator signal sequence fusion proteins or ubiquitin-conjugated antigens induce sustained protective immunity in a mouse model of pulmonary tuberculosis. Infect Immun 2002;70:292–302. [69] Morris S, Kelley C, Howard A, Li Z, Collins F. The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine 2000;18:2155–63.