Vaccine 31 (2013) 6065–6071
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Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii Qinlei Yu a,b,1 , Xiangsheng Huang a,c,1 , Pengtao Gong a,∗ , Qian Zhang a , Jianhua Li a , Guocai Zhang a , Ju Yang a , He Li a , Nan Wang b , Xichen Zhang a,∗ a
Institute of Zoonosis, College of Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, China Jilin Provincial Animal Disease Control Center, 4510 Xi’an Road, Changchun 130062, China c Institute for Tropical Medicine, University of Tübingen, Wilhelmstrasse 27, Tübingen 72074, Germany b
a r t i c l e
i n f o
Article history: Received 21 May 2013 Received in revised form 14 September 2013 Accepted 3 October 2013 Available online 29 October 2013 Keywords: T. gondii Cyclophilin rBCG BALB/c
a b s t r a c t The investigation of Toxoplasma gondii virulence factors can elucidate the immunopathology of T. gondii infection and identify potential candidates for effective human vaccines. The adjuvant is an important component of an effective vaccine. In this study, attenuated Mycobacterium bovis was used as a live vaccine vector with both antigen and adjuvant characteristics. Following amplification of the T. gondii cyclophilin gene, the shuttle expression plasmid pMV261-TgCyP and integrative expression plasmid pMV361-TgCyP were constructed, and their expression was stimulated after transfection into BCG. Both recombinant plasmids were highly immunogenic. Greater proliferation of CD4+ and CD8+ T cells was observed in the rBCG-vaccinated groups compared to the control groups. The levels of Th1-type IFN-␥, IL-2 and IL-12 were significantly increased following immunisation with the rBCG vaccines via the i.v. or oral route, which indicated that catalytic activity against T. gondii infection was generated in the mice. rBCGpMV361-TgCyP i.v. inoculation resulted in a higher protection efficiency, as demonstrated by the increased survival time and survival rate (17%) of BALB/c mice. The present study demonstrates that a BCG vector expressing a target antigen, TgCyP, represent an alternative system for the production of effective vaccines to prevent toxoplasmosis. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii (T. gondii) is a widespread opportunistic protozoan (phylum Apicomplexa) pathogen. T. gondii replicates within the nucleated cells of warm-blooded animals and causes severe toxoplasmosis in humans and livestock [1,2]. T. gondii infection is normally asymptomatic or causes a mild, flu-like illness. However, it can be lethal in patients with acquired immunodeficiencies or those who have undergone organ transplantation [3,4]. Treatment of toxoplasmosis with chemical drugs is difficult due to toxic side effects. Although there are live attenuated vaccines available for veterinary use, they are expensive and cause side effects, and their use is limited. Nevertheless, a safe, cheap and efficient vaccine against T. gondii is the appropriate solution for controlling toxoplasmosis [5]. T. gondii modifies its host’s behaviour dexterously, which may allow the pathogen to escape from phagocytosis by the host’s
∗ Corresponding authors. Tel.: +86 139 04310460; fax: +86 431 87981351. E-mail addresses: [email protected], [email protected], [email protected] (X. Zhang). 1 These authors contributed equally to this work. 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.10.015
immune cells. During T. gondii invasion, fast-replicating tachyzoites are replaced by slowly multiplying bradyzoites (cysts formed in tissues), which may be one mechanism through which T. gondii evades host immunity. The cysts establish a life-long chronic infection and prevent host death. Pressure from the immune response can cause T. gondii stage conversion, which involves many genes [6–8]. Some of these genes, such as MAG1, GRA1, ROP1 and MIC1, regulate tachyzoite and bradyzoite differentiation [7,8]. A T. gondii ROP1 DNA vaccine was shown to induce a Th1-specific immune response in BALB/c mice and sheep [9,10], and MIC1 and MIC3 knockout T. gondii-vaccinated ewes are protected from T. gondii-induced abortion [11]. Therefore, identifying and characterising these regulatory genes may elucidate the interactions between T. gondii and its vertebrate hosts and may aid in the development of vaccines against toxoplasmosis. T. gondii cyclophilin (TgCyP) is released by extracellular tachyzoites, which is vital for the conversion of the T. gondii life cycle because TgCyP induces nitric oxide (NO) production [12]. The production of NO, TNF-␣ and IL-12 is induced by TgCyP through interaction with cysteine-cysteine chemokine receptor 5 (CCR5) in dendritic cells and macrophages [12,13]. Furthermore, CCR5 depletion causes fatalities in infected mice due to uncontrolled T. gondii replication [14]. Neospora cyclophilin (NcCyP), which is closely related to TgCyP, may control acute neosporosis [15]. A TgCyP DNA
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vaccine has been shown to promote protective immunity against T. gondii infection in BALB/c mice [16]. Thus, TgCyP is an excellent candidate for a vaccine. Bacillus Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis (M. bovis). After more than 200 passages in vitro, several gene complexes were observed to be lost or rearranged in BCG [17]. BCG is uniquely suited to be used as a vaccine because it is stable, safe, easy to apply and cost effective. Recombinant BCG (rBCG) has been widely investigated due to its excellent adjuvant properties [18]. Antigens from parasites, bacteria and viruses that are expressed in BCG have been shown to induce humoural and cellular immune responses in several animals, including mice and guinea pigs [19,20]. When expressed in rBCG, the Leishmania chagasi (L. chagasi) LCR1 antigen induces the production of IFN-gamma (IFN-␥) and protects against virulent L. chagasi challenge in mice [21]. rBCG expressing the T. gondii GRA1 antigen induces specific cell-mediated responses and results in partial protection in sheep [22]. Thus, BCG is a potential vaccine vector for the expression and presentation of T. gondii antigens [23]. The promoters in the mycobacterium shuttle vector and the integrative vector can modulate the expression of foreign genes in mycobacteria. In the present study, we selected the pMV261 and pMV361 vectors, which contain efficient promoters from the heat shock protein gene hsp60 [18,24]. pMV261 and pMV361 share many elements, and their expression is comparable. The integrative plasmid pMV361 is more stable during stress responses [18]. In the present study, we constructed two rBCG-carrying plasmids (rBCGpMV261TgCyP and rBCGpMV361-TgCyP) that express the TgCyP antigen and evaluated the humoural and cellular immune responses produced during T. gondii infection. The efficiency of protection was determined by challenge with a highly virulent T. gondii RH strain following intravenous (i.v.) or oral vaccination in BALB/c mice.
TTA CTC CAA CAA ACC AAT GTC CGT-3 . The forward and reverse primers for the pMV361 vector contained HindIII and ClaI restriction sites, respectively: forward primer, 5 -AAG CTT ATG AAG CTC GTG CTG TTT TTC CT-3 ; reverse primer, 5 -ATC GAT TTA CTC CAA CAA ACC AAT GTC CGT-3 . The applied PCR conditions were as follows: pre-denaturation at 94 ◦ C for 30 s, followed by 30 cycles of denaturing at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min. The amplified TgCyP DNA fragment was sub-cloned into the shuttle vector pMV261 or the integration vector pMV361 to form the plasmids pMV261-TgCyP and pMV361-TgCyP, respectively. Plasmid sequences were confirmed via PCR and double restriction enzyme digestion. The concentrations of the recombinant plasmids were determined via spectrophotometry (optical density at 260 nm). The recombinant plasmids pMV261-TgCyP and pMV361-TgCyP were electroporated into BCG, followed by kanamycin selection, as previously described [23,28]. Briefly, M. bovis BCG was cultivated to the log growth phase and collected via centrifugation. The BCG bacteria were next mixed with recombinant plasmids. And the electroporation was performed with a Gene Pulser (BTX Instrument Division, Holliston, MA, USA). The bacteria were subsequently cultured on Middlebrook 7H10 medium. After 4 weeks, single colonies were picked and transferred to liquid medium for further study. Following, mass bacteria were harvested and the protein concentration was determined via a BCA protein assay, and western blotting was performed as described previously [28]. Briefly, extracts from BCG and rBCGpMV261-TgCyP- and rBCGpMV361TgCyP-transfected cells were separated in 12% SDS–PAGE gels, after which the proteins were electro-transferred to a PVDF membrane, and the expression of TgCyP was detected with an anti-T. gondii tachyzoite polyclonal antibody (1:1000) and HRP-conjugated goat anti-mouse IgG (1:500).
2. Materials and methods
2.3. Immunisation with rBCG and T. gondii challenge in BALB/c mice
2.1. Preparation of a polyclonal antibody specific to T. gondii tachyzoites T. gondii (RH strain) tachyzoites were propagated via serial intraperitoneal passaging through 6- to 8-week-old Kunming mice every 2 months (1 × 103 tachyzoites/mouse). The tachyzoites were harvested from the supernatant of collected peritoneal fluid following centrifugation (600 × g for 10 min) and washed with 0.01 M PBS (pH 7.2). All experimental procedures were conducted according to the guidelines of the Jilin University Experimental Animal Center. Similar to previously described standard procedures for polyclonal antibody preparation [23,25,26], purified T. gondii tachyzoites (5 × 108 ) were used to prepare toxoplasma lysate antigen (TLA). Briefly, each BALB/c mouse was injected every two weeks with 100 g of TLA, which was emulsified with an equal volume of Freund’s complete adjuvant for the first injection and an equal volume of Freund’s incomplete adjuvant for the second and third injections. Six weeks later, the anti-T. gondii tachyzoite polyclonal antibody produced was collected and used for western blotting. 2.2. pMV261-TgCyP and pMV361-TgCyP plasmid construction and expression in BCG T. gondii tachyzoite cDNA was synthesised via reverse transcriptase PCR using an oligo dT primer, as described previously [27], and the obtained cDNA was used as a template to amplify the coding region of TgCyP through PCR (Biometra, Germany). The TgCyP primers for the pMV261 vector were designed to include EcoRI and SalI restriction sites, as follows: forward primer, 5 -GAA TTC ATG AAG CTC GTG CTG TTT TTC CT-3 ; reverse primer, 5 -GTC GAC
Positive rBCGpMV261-TgCyP and rBCGpMV361-TgCyP bacterial pellets were collected and washed twice with 0.9% saline for further immunisation. 6- to 8-week-old female BALB/c mice were randomly divided into six groups (twelve mice per group). rBCGpMV261-TgCyP and rBCGpMV361-TgCyP (106 CFU) were used to immunise the mice in two different manners (through an i.v. or oral route). Mice receiving BCG alone (106 CFU, i.v.) or PBS (0.1 ml, i.v.) were used as negative controls. The vaccination interval was 2 weeks, and all groups were vaccinated three times (on days 0, 14 and 28). Blood was collected from each group via the venous plexus of the tail prior to each vaccination and stored at −20 ◦ C for enzyme-linked immunosorbent assays (ELISAs). Two weeks after the third vaccination, six BALB/c mice were selected randomly from each group and challenged intraperitoneally (i.p.) with 103 highly virulent T. gondii RH strain tachyzoites. All mice were observed twice daily, and their survival times were recorded. 2.4. Humoral immune responses and cellular immune responses induced by rBCGs in BALB/c mice An indirect ELISA was performed to evaluate the humoural immune response stimulated by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP, as described previously [26]. Briefly, 96well microtitre plates were coated with crude T. gondii tachyzoite antigens (10 g/ml). On the next day, 5% bovine serum albumin (BSA; 100 l/well) was added to block non-specific binding. The plates were next incubated with the collected mouse sera (1:3200 in 1% BSA-PBS). Later, the plates were incubated with an HRP-labelled goat anti-mouse IgG antibody (1:3000, BOSTER,
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China). After washing with PBST, a substrate solution was added. Stop solution (2 M H2 SO4 ) was added to end the reaction, and the optical density values were read at A450 . Two weeks after the final vaccination, the spleens of six mice per group were removed, gently squeezed and passed through a 200-mesh sieve with PBS. The supernatants were then harvested via centrifugation and diluted 1:1 with lymphocyte separation medium. Following centrifugation, the cell suspensions were collected from the middle layer of the tube, after which fluorescein isothiocyanate-labelled anti-CD4+ and anti-CD8+ T cell antibodies were added, and the cells were stored in the dark for 40 min. After washing with PBS, the CD4+ and CD8+ T cell populations were analysed via flow cytometry. The double antibody sandwich method was used to evaluate the expression of IL-2, IL-4, IL-12 and IFN-␥ in the sera collected from immunised BALB/c mice, per the manufacturer’s protocol (Jiamay Biotech, Beijing, China). A 96-well microplate was coated with the specific capture antibody and incubated overnight. On the second day, the plate was washed and blocked with 5% BSA, and sera samples were added, followed by incubation at room temperature for 2 h and subsequent application of an HRP-labelled detection antibody. Finally, the substrate TMB and an H2 SO4 stop solution were applied for colour development, and the optical density values were read at A450 . 2.5. Statistical analysis Statistical analysis was performed using SPSS 14.0 software for analysis of variance (ANOVA) and Duncan’s multiple range test. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Expression of the recombinant plasmids pMV261-TgCyP and pMV361-TgCyP in BCG The coding region of TgCyP was amplified via RT-PCR and cloned into both a shuttle expression vector, pMV261, and an integration expression vector, pMV361. The constructed plasmids were designated pMV261-TgCyP and pMV361-TgCyP and were verified by PCR
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Fig. 1. Western blotting assay of the expression of recombinant TgCyP protein in rBCG. Anti-T. gondii tachyzoite polyclonal antibody and HRP goat anti-mouse IgG were used to detect the specific expression of target proteins. N: negative control; 1: rBCGpMV361-TgCyP; 2: rBCGpMV261-TgCyP.
and sequencing. Positive clones were selected based on kanamycin resistance, and the selected rBCG bacteria were stimulated at 45 ◦ C to induce the expression of the target proteins. Western blotting showed that these recombinant plasmids were successfully expressed in vitro and were highly immunogenic (Fig. 1). 3.2. Humoral immune responses induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice Specific antibody responses against T. gondii tachyzoites were detected in all of the rBCG-vaccinated BALB/c mice through ELISA. Gradually increased IgG levels were observed in all of the rBCG- and BCG-vaccinated groups following each immunisation. From 2 to 6 weeks, all of the groups immunised with rBCG via the oral or i.v. route showed significantly higher IgG levels than the PBS groups (P < 0.001). Although the IgG level induced by BCG was elevated, it was still significantly lower than in the rBCG-vaccinated groups (P < 0.001; Fig. 2). 3.3. Cellular immune response induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice Splenocytes were harvested from mice and separated using lymphocyte separation medium. Flow cytometry analysis showed that the population of CD4+ T lymphocytes was significantly expanded in the rBCGpMV261-TgCyP orally vaccinated and the rBCGpMV261-TgCyP and rBCGpMV361-TgCyP i.v.-vaccinated groups. Furthermore, the percentage of CD8+ T lymphocytes increased gradually following rBCG immunisation. Thus, the i.v.
Fig. 2. Humoral immune responses induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP vaccines. BALB/c mice were immunized via either i.v. or oral route with rBCG vaccines. Serum were collected from 12 randomly mice of each group in 0 week, 2 weeks, 4 weeks and 6 weeks and measured for specific anti-T. gondii TLA antibodies by indirect ELISA. Data are represents by the mean OD ± S.E. (n = 12). Statistically significant differences are marked with the asterisk (compared with PBS group, * P < 0.05, **P < 0.01, ***P < 0.001).
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Table 1 Percentages of CD4+ and CD8* T cells in splenocytes from immunized BALB/c mice. Groups rBCGpMV261-TgCyPi.v. rBCGpMV261-TgCyP oral rBCGpMV361-TgCyPi.v. rBCGpMV361-TgCyP oral BCG j.v. PBS i.v.
CD4+ (%) 35.2 29.1 32.3 28.7 26.5 22.7
± ± ± ± ± ±
3.4. Protective efficacy of rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice
CD8*+ (%) *
0.28 0.25* 0.28* 0.16 0.18 0.21
18.32 15.60 17.1 15.4 15.8 14.5
± ± ± ± ± ±
0.16 0.14 0.12 0.15 0.12 0.19
Note: Data are represented by the Mean ± S.E. (n = 6). * Groups marked with asterisk are significantly different from the PBS control group (* P < 0.05).
route was more effective than the oral route in inducing a T. gondiispecific immune response (Table 1). Additionally, the levels of Th1-type IFN-␥, IL-2 and IL-12 and Th2-type IL-4 in vaccinated BALB/c mice were evaluated using a double antibody sandwich ELISA method. The ELISA results indicated higher levels of IFN-␥, IL-2 and IL-12 in the rBCGvaccinated groups, while the PBS and BCG groups showed only slight alterations in the levels of these cytokines (Fig. 3). Interestingly, intravenous injection induced the highest levels of IFN-␥, IL-2 and IL-12 observed in BALB/c mice, even compared to oral vaccination, and there was a significant difference detected compared to the control groups (P < 0.001). There was no difference between the experimental groups and control groups regarding the production of Th2-type IL-4 (P > 0.05). Furthermore, rapid production of IFN␥ early in the immune response, prior to the production IL-2 and IL-12, was observed in the rBCG groups.
Two weeks after the last vaccination, six mice were selected randomly from each group and challenged intraperitoneally with 103 highly virulent T. gondii RH strain tachyzoites. Overall, the rBCGvaccinated groups exhibited a higher protective efficacy than the control groups. The survival rate of the rBCGpMV361-TgCyP i.v.vaccinated group was 17% after 14 days, while the mice in all other groups expired prior to this time point (Fig. 4). Nevertheless, the rBCG-vaccinated group showed a prolonged survival time compared to the control groups. Thus, the i.v. route was superior to the oral route, as similar results were observed in terms of anti-T. gondii antibody production and the cellular response. Overall, the rBCGpMV261-TgCyP and rBCGpMV361-TgCyP vaccines limited T. gondii infection in BALB/c mice. 4. Discussion The present study was performed to determine the efficiency of recombinant BCG vaccines expressing the T. gondii cyclophilin gene in BALB/c mice. A significant and specific immune response was induced by the rBCG vaccines, administered either via i.v. or oral injection. Although the BCG vaccines were originally designed and tested through oral injection, the i.v. route is presumed to be the best way to administer rBCG vaccines. This study investigated the efficiency of i.v. injection of rBCG vaccines in mice. A protection efficiency of 17% was determined for the rBCGpMV361-TgCyP vaccine in BALB/c mice following challenge with T. gondii tachyzoites. As
Fig. 3. Cytokine production induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP immunized mice. The serum of immunized mice was collected in 0 week, 2 weeks, 4 weeks and 6 weeks, and evaluated by ELISA for the Th1 and Th2 type cytokines production. (a) IFN-␥ production; (b) IL-2 production; (c) IL-4 production; (d) IL-12 production. Data are showed as mean ± S.D. (n = 6). Statistically significant differences are indicated by the asterisk (compared with PBS group, *P < 0.05, **P < 0.01, ***P < 0.001).
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Fig. 4. Survival rate of rBCGpMV261-TgCyP and rBCGpMV361-TgCyP immunized BALB/c mice after T. gondii challenge. Each mouse was given intraperitoneally with 103 T. gondii RH strain tachyzoites at 2 weeks after the last immunization (n = 6). Mice were monitored daily and survival times were recorded. The survival rate of rBCGpMV361TgCyP vaccinated mice can reach to 17%.
previously reported, the genetically stable integrative vector pMV361 was more efficient than the shuttle vector pMV-261 [29]. TgCyP has been indicated as a potential candidate gene for the development of an anti-T. gondii DNA vaccine [16]. This study was the first to express the T. gondii cyclophilin gene in exogenous attenuated Mycobacterium bovis. Overall, a significantly higher specific anti-T. gondii immune response was induced in rBCG vaccinated groups. Although the attained protection efficiency was low, BCG acted both as an adjuvant and a vehicle in eliminating T. gondii infection, and the TgCyP antigen played the dominant role in protection. BCG bacteria survive and replicate inside antigen-presenting cells (APCs), such as macrophages and dendritic cells. These APCs present BCG or heterologous antigens to initiate an immune response in mice [30]. rBCG induces both humoural and cellular immune responses against many heterologous antigens, including those from parasites, bacteria and viruses [31]. Similar to the results of a previous study, the TgCyP antigen was successfully expressed in BCG in the present work. In developing an rBCG vaccine, the main concern is the stability and expression efficiency of rBCG in vitro and in vivo. Multicopy plasmids can increase the expression level in an integrative BCG vector [31]. For some antigens, such as HIV-1 gp120, single copy integrative vectors are preferred, as high copy numbers may be lethal (e.g., 5 copies in pMV261) [18]. Because the pMV261 and pMV361 vectors utilise the highly transcript hsp60 promoter, they may be advantageous for rBCG vaccine development [18,32]. The pBMX vector, containing the hsp60 promoter, increases the expression of foreign antigens and elicits high antibody responses in BALB/c mice [33]. Similarly, a high level of specific anti-T. gondii IgG antibody production was observed in this study. Inoculation via the i.v. route results in the generation of specific antibodies and splenocyte proliferation in mice [32,34]. Mice immunised with rBCG via the oral route exhibit strong cellular and IgA responses to -galactosidase [35]. Administration through both the i.v. and oral routes in the present study induced a strong immune response against the BCG-expressed TgCyP antigen. Stronger anti-T. gondii immune responses were produced in the rBCG groups, with the greatest antibodies release being detected in the rBCGpMV361-TgCyP i.v. group. Administration via the i.v. route induced more antibody production compared to the oral route. I.v. immunisation also elicits a greater specific response than subcutaneous or intranasal inoculation against Schistosoma mansoni (S. mansoni) using Sm28GST [34]. This result may confirm the speculation that foreign antigens initiate different immune responses depending on the mode of immunisation [34]. Oral inoculation also significantly elevated antibody levels and cellular responses in
BALB/c mice. Although the immune responses to oral BCG vaccines are controversial, they are the best way for appropriate antigens to be taken up by the human gut [36–40]. As BCG induces shortterm resistance to S. mansoni, specific antibody responses should be stimulated by BCG, such as the increased antibody response observed in this study [41]. Recombinant proteins expressed by rBCG elicit more robust antibody responses than BCG antigen alone, which may persist much longer in mice [18]. In the present study, antibody production gradually increased in the rBCG groups, and a significant difference was observed even following the first inoculation, confirming that a single immunisation induces a strong immune response [18]. Likewise, specific immune responses were shown to be induced by T. gondii ROP2 expressed in BCG [23]. At longer times post-inoculation, the detected IgG levels were much higher, suggesting that rBCG induces a more efficient boost in foreign antigens and a stronger immune response. Green fluorescent protein expressed from the vector pMV261 is clearly visible inside human macrophages [42]. In general, cytosolic antigens are processed through the MHC class I pathway to induce CD8+ T cell production [43,44]. After BCG bacteria are killed, the released antigens are delivered to the phagolysosome and processed via the MHC class II pathway, predominantly stimulating CD4+ T cells [45]. The significantly increased production of CD4+ T cells observed in rBCG-vaccinated groups supports this hypothesis. The greater number of CD4+ T cells produced confirmed their dominant role in the rBCG-induced cellular immune response, while the increase in CD8+ T cells may have resulted from the initial cytotoxicity of BCG. CD4+ and CD8+ T cells are involved in the protection elicited by an rBCG-expressing membrane-associated protein [46]. rBCG inoculation enhances the Th1 response, as indicated by IFN-␥ levels, while suppressing the production of Th2-type IL-10 and IL-4 [47,48]. Th2-type IL-4, which is mainly involved in allergic reactions and asthma, cannot modulate BCG stimulation [49]. IL-12 is involved in the differentiation of naive T cells into Th1 cells and acts together with IL-18 to induce cell-mediated immunity following infection with microbial products such as lipopolysaccharide [50]. The secretion of IL-2 and IL-18 by the BCG strain enhances long-lasting Th1 phenotype-associated cellular immunity in mice [51–54]. IL-2 and IFN-␥ may modify the immune responses to BCG antigens [51,52,55]. Significantly high IFN-␥ levels have been observed in association with rBCG vaccines expressing the L. chagasi LCR1 antigen, S. mansoni Sm14 antigen and Leptospira interrogans LipL32 antigen [21,56,57]. In the present study, the levels of Th1-type IFN-␥, IL-2 and IL-12 were highly elevated following the administration of rBCG or BCG, while rBCG did not alter the
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production of Th2-type IL-4, suggesting that the Th1 response is the dominant cellular response to the BCG-expressed TgCyP antigen. Mice vaccinated with rBCG expressing membrane-associated proteins are better protected from challenge with Borrelia burgdorferi, Listeria or pneumococcal infection than mice receiving vaccines expressing cytosolic proteins [46,58,59]. Immunisation with rBCG expressing the C-terminal fragment of MSP-1 protects mice from Plasmodium yoelii challenge [60]. In the present study, we observed prolonged survival times in BALB/c mice vaccinated with rBCG. I.v. administration of the rBCGpMV361-TgCyP vaccine resulted in a protection efficiency of 17%. Higher humoural or cellular immune responses can be induced by linking a specific targeting sequence to a foreign antigen, such as the Mycobacterium tuberculosis 19 kDa lipoprotein, PspA signal sequence or mycobacterial ␣ antigen, compared to expression in the cytoplasm [39,46,59,61]. Thus, TgCyP delivered via BCG and expressed in the cytoplasm may confer limited protection against T. gondii challenge. We demonstrated that a BCG-expressing TgCyP vaccine was relatively effective in protecting BALB/c mice against T. gondii infection. Many factors, such as the promoter, site of expression, route of administration, dose administered and animal model involved, are of concern for rBCG vaccine development and for building the most effective BCG expression system for certain antigens. These considerations will prompt further studies investigating potential vaccine candidate antigens. Conflict of interest The authors have no competing interests. Acknowledgements This work was supported by a grant from the Special Fund for Agro-scientific Research in the Public Interest in China (no. 20130304). References [1] McCabe R, Remington JS. Toxoplasmosis: the time has come. The New England Journal of Medicine 1988;318:313–5. [2] Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clinical Microbiology and Infection: The Official Publication of The European Society of Clinical Microbiology and Infectious Diseases 2002;8:634–40. [3] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965–76. [4] Dubey JP, Jones JL. Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology 2008;38:1257–78. [5] Innes EA, Vermeulen AN. Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology 2006;133:S145–68. [6] Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews 1998;11:267–99. [7] Cleary MD, Singh U, Blader IJ, Brewer JL, Boothroyd JC. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryotic Cell 2002;1:329–40. [8] Radke JR, Behnke MS, Mackey AJ, Radke JB, Roos DS, White MW. The transcriptome of Toxoplasma gondii. BMC Biology 2005;3:26. [9] Quan JH, Chu JQ, Ismail HA, Zhou W, Jo EK, Cha GH, et al. Induction of protective immune responses by a multiantigenic DNA vaccine encoding GRA7 and ROP1 of Toxoplasma gondii. Clinical and Vaccine Immunology: CVI 2012;19:666–74. [10] Hiszczynska-Sawicka E, Li H, Xu JB, Holec-Gasior L, Kur J, Sedcole R, et al. Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154. Veterinary Parasitology 2011;183:72–8. [11] Mevelec MN, Ducournau C, Bassuny Ismael A, Olivier M, Seche E, Lebrun M, et al. Mic1-3 knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep. Veterinary Research 2010;41:49. [12] Ibrahim HM, Bannai H, Xuan X, Nishikawa Y. Toxoplasma gondii cyclophilin 18mediated production of nitric oxide induces Bradyzoite conversion in a CCR5dependent manner. Infection and Immunity 2009;77:3686–95. [13] Aliberti J, Valenzuela JG, Carruthers VB, Hieny S, Andersen J, Charest H, et al. Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells. Nature Immunology 2003;4:485–90.
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Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii Qinlei Yu a,b,1 , Xiangsheng Huang a,c,1 , Pengtao Gong a,∗ , Qian Zhang a , Jianhua Li a , Guocai Zhang a , Ju Yang a , He Li a , Nan Wang b , Xichen Zhang a,∗ a
Institute of Zoonosis, College of Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, China Jilin Provincial Animal Disease Control Center, 4510 Xi’an Road, Changchun 130062, China c Institute for Tropical Medicine, University of Tübingen, Wilhelmstrasse 27, Tübingen 72074, Germany b
a r t i c l e
i n f o
Article history: Received 21 May 2013 Received in revised form 14 September 2013 Accepted 3 October 2013 Available online 29 October 2013 Keywords: T. gondii Cyclophilin rBCG BALB/c
a b s t r a c t The investigation of Toxoplasma gondii virulence factors can elucidate the immunopathology of T. gondii infection and identify potential candidates for effective human vaccines. The adjuvant is an important component of an effective vaccine. In this study, attenuated Mycobacterium bovis was used as a live vaccine vector with both antigen and adjuvant characteristics. Following amplification of the T. gondii cyclophilin gene, the shuttle expression plasmid pMV261-TgCyP and integrative expression plasmid pMV361-TgCyP were constructed, and their expression was stimulated after transfection into BCG. Both recombinant plasmids were highly immunogenic. Greater proliferation of CD4+ and CD8+ T cells was observed in the rBCG-vaccinated groups compared to the control groups. The levels of Th1-type IFN-␥, IL-2 and IL-12 were significantly increased following immunisation with the rBCG vaccines via the i.v. or oral route, which indicated that catalytic activity against T. gondii infection was generated in the mice. rBCGpMV361-TgCyP i.v. inoculation resulted in a higher protection efficiency, as demonstrated by the increased survival time and survival rate (17%) of BALB/c mice. The present study demonstrates that a BCG vector expressing a target antigen, TgCyP, represent an alternative system for the production of effective vaccines to prevent toxoplasmosis. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii (T. gondii) is a widespread opportunistic protozoan (phylum Apicomplexa) pathogen. T. gondii replicates within the nucleated cells of warm-blooded animals and causes severe toxoplasmosis in humans and livestock [1,2]. T. gondii infection is normally asymptomatic or causes a mild, flu-like illness. However, it can be lethal in patients with acquired immunodeficiencies or those who have undergone organ transplantation [3,4]. Treatment of toxoplasmosis with chemical drugs is difficult due to toxic side effects. Although there are live attenuated vaccines available for veterinary use, they are expensive and cause side effects, and their use is limited. Nevertheless, a safe, cheap and efficient vaccine against T. gondii is the appropriate solution for controlling toxoplasmosis [5]. T. gondii modifies its host’s behaviour dexterously, which may allow the pathogen to escape from phagocytosis by the host’s
∗ Corresponding authors. Tel.: +86 139 04310460; fax: +86 431 87981351. E-mail addresses: [email protected], [email protected], [email protected] (X. Zhang). 1 These authors contributed equally to this work. 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.10.015
immune cells. During T. gondii invasion, fast-replicating tachyzoites are replaced by slowly multiplying bradyzoites (cysts formed in tissues), which may be one mechanism through which T. gondii evades host immunity. The cysts establish a life-long chronic infection and prevent host death. Pressure from the immune response can cause T. gondii stage conversion, which involves many genes [6–8]. Some of these genes, such as MAG1, GRA1, ROP1 and MIC1, regulate tachyzoite and bradyzoite differentiation [7,8]. A T. gondii ROP1 DNA vaccine was shown to induce a Th1-specific immune response in BALB/c mice and sheep [9,10], and MIC1 and MIC3 knockout T. gondii-vaccinated ewes are protected from T. gondii-induced abortion [11]. Therefore, identifying and characterising these regulatory genes may elucidate the interactions between T. gondii and its vertebrate hosts and may aid in the development of vaccines against toxoplasmosis. T. gondii cyclophilin (TgCyP) is released by extracellular tachyzoites, which is vital for the conversion of the T. gondii life cycle because TgCyP induces nitric oxide (NO) production [12]. The production of NO, TNF-␣ and IL-12 is induced by TgCyP through interaction with cysteine-cysteine chemokine receptor 5 (CCR5) in dendritic cells and macrophages [12,13]. Furthermore, CCR5 depletion causes fatalities in infected mice due to uncontrolled T. gondii replication [14]. Neospora cyclophilin (NcCyP), which is closely related to TgCyP, may control acute neosporosis [15]. A TgCyP DNA
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vaccine has been shown to promote protective immunity against T. gondii infection in BALB/c mice [16]. Thus, TgCyP is an excellent candidate for a vaccine. Bacillus Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis (M. bovis). After more than 200 passages in vitro, several gene complexes were observed to be lost or rearranged in BCG [17]. BCG is uniquely suited to be used as a vaccine because it is stable, safe, easy to apply and cost effective. Recombinant BCG (rBCG) has been widely investigated due to its excellent adjuvant properties [18]. Antigens from parasites, bacteria and viruses that are expressed in BCG have been shown to induce humoural and cellular immune responses in several animals, including mice and guinea pigs [19,20]. When expressed in rBCG, the Leishmania chagasi (L. chagasi) LCR1 antigen induces the production of IFN-gamma (IFN-␥) and protects against virulent L. chagasi challenge in mice [21]. rBCG expressing the T. gondii GRA1 antigen induces specific cell-mediated responses and results in partial protection in sheep [22]. Thus, BCG is a potential vaccine vector for the expression and presentation of T. gondii antigens [23]. The promoters in the mycobacterium shuttle vector and the integrative vector can modulate the expression of foreign genes in mycobacteria. In the present study, we selected the pMV261 and pMV361 vectors, which contain efficient promoters from the heat shock protein gene hsp60 [18,24]. pMV261 and pMV361 share many elements, and their expression is comparable. The integrative plasmid pMV361 is more stable during stress responses [18]. In the present study, we constructed two rBCG-carrying plasmids (rBCGpMV261TgCyP and rBCGpMV361-TgCyP) that express the TgCyP antigen and evaluated the humoural and cellular immune responses produced during T. gondii infection. The efficiency of protection was determined by challenge with a highly virulent T. gondii RH strain following intravenous (i.v.) or oral vaccination in BALB/c mice.
TTA CTC CAA CAA ACC AAT GTC CGT-3 . The forward and reverse primers for the pMV361 vector contained HindIII and ClaI restriction sites, respectively: forward primer, 5 -AAG CTT ATG AAG CTC GTG CTG TTT TTC CT-3 ; reverse primer, 5 -ATC GAT TTA CTC CAA CAA ACC AAT GTC CGT-3 . The applied PCR conditions were as follows: pre-denaturation at 94 ◦ C for 30 s, followed by 30 cycles of denaturing at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min. The amplified TgCyP DNA fragment was sub-cloned into the shuttle vector pMV261 or the integration vector pMV361 to form the plasmids pMV261-TgCyP and pMV361-TgCyP, respectively. Plasmid sequences were confirmed via PCR and double restriction enzyme digestion. The concentrations of the recombinant plasmids were determined via spectrophotometry (optical density at 260 nm). The recombinant plasmids pMV261-TgCyP and pMV361-TgCyP were electroporated into BCG, followed by kanamycin selection, as previously described [23,28]. Briefly, M. bovis BCG was cultivated to the log growth phase and collected via centrifugation. The BCG bacteria were next mixed with recombinant plasmids. And the electroporation was performed with a Gene Pulser (BTX Instrument Division, Holliston, MA, USA). The bacteria were subsequently cultured on Middlebrook 7H10 medium. After 4 weeks, single colonies were picked and transferred to liquid medium for further study. Following, mass bacteria were harvested and the protein concentration was determined via a BCA protein assay, and western blotting was performed as described previously [28]. Briefly, extracts from BCG and rBCGpMV261-TgCyP- and rBCGpMV361TgCyP-transfected cells were separated in 12% SDS–PAGE gels, after which the proteins were electro-transferred to a PVDF membrane, and the expression of TgCyP was detected with an anti-T. gondii tachyzoite polyclonal antibody (1:1000) and HRP-conjugated goat anti-mouse IgG (1:500).
2. Materials and methods
2.3. Immunisation with rBCG and T. gondii challenge in BALB/c mice
2.1. Preparation of a polyclonal antibody specific to T. gondii tachyzoites T. gondii (RH strain) tachyzoites were propagated via serial intraperitoneal passaging through 6- to 8-week-old Kunming mice every 2 months (1 × 103 tachyzoites/mouse). The tachyzoites were harvested from the supernatant of collected peritoneal fluid following centrifugation (600 × g for 10 min) and washed with 0.01 M PBS (pH 7.2). All experimental procedures were conducted according to the guidelines of the Jilin University Experimental Animal Center. Similar to previously described standard procedures for polyclonal antibody preparation [23,25,26], purified T. gondii tachyzoites (5 × 108 ) were used to prepare toxoplasma lysate antigen (TLA). Briefly, each BALB/c mouse was injected every two weeks with 100 g of TLA, which was emulsified with an equal volume of Freund’s complete adjuvant for the first injection and an equal volume of Freund’s incomplete adjuvant for the second and third injections. Six weeks later, the anti-T. gondii tachyzoite polyclonal antibody produced was collected and used for western blotting. 2.2. pMV261-TgCyP and pMV361-TgCyP plasmid construction and expression in BCG T. gondii tachyzoite cDNA was synthesised via reverse transcriptase PCR using an oligo dT primer, as described previously [27], and the obtained cDNA was used as a template to amplify the coding region of TgCyP through PCR (Biometra, Germany). The TgCyP primers for the pMV261 vector were designed to include EcoRI and SalI restriction sites, as follows: forward primer, 5 -GAA TTC ATG AAG CTC GTG CTG TTT TTC CT-3 ; reverse primer, 5 -GTC GAC
Positive rBCGpMV261-TgCyP and rBCGpMV361-TgCyP bacterial pellets were collected and washed twice with 0.9% saline for further immunisation. 6- to 8-week-old female BALB/c mice were randomly divided into six groups (twelve mice per group). rBCGpMV261-TgCyP and rBCGpMV361-TgCyP (106 CFU) were used to immunise the mice in two different manners (through an i.v. or oral route). Mice receiving BCG alone (106 CFU, i.v.) or PBS (0.1 ml, i.v.) were used as negative controls. The vaccination interval was 2 weeks, and all groups were vaccinated three times (on days 0, 14 and 28). Blood was collected from each group via the venous plexus of the tail prior to each vaccination and stored at −20 ◦ C for enzyme-linked immunosorbent assays (ELISAs). Two weeks after the third vaccination, six BALB/c mice were selected randomly from each group and challenged intraperitoneally (i.p.) with 103 highly virulent T. gondii RH strain tachyzoites. All mice were observed twice daily, and their survival times were recorded. 2.4. Humoral immune responses and cellular immune responses induced by rBCGs in BALB/c mice An indirect ELISA was performed to evaluate the humoural immune response stimulated by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP, as described previously [26]. Briefly, 96well microtitre plates were coated with crude T. gondii tachyzoite antigens (10 g/ml). On the next day, 5% bovine serum albumin (BSA; 100 l/well) was added to block non-specific binding. The plates were next incubated with the collected mouse sera (1:3200 in 1% BSA-PBS). Later, the plates were incubated with an HRP-labelled goat anti-mouse IgG antibody (1:3000, BOSTER,
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China). After washing with PBST, a substrate solution was added. Stop solution (2 M H2 SO4 ) was added to end the reaction, and the optical density values were read at A450 . Two weeks after the final vaccination, the spleens of six mice per group were removed, gently squeezed and passed through a 200-mesh sieve with PBS. The supernatants were then harvested via centrifugation and diluted 1:1 with lymphocyte separation medium. Following centrifugation, the cell suspensions were collected from the middle layer of the tube, after which fluorescein isothiocyanate-labelled anti-CD4+ and anti-CD8+ T cell antibodies were added, and the cells were stored in the dark for 40 min. After washing with PBS, the CD4+ and CD8+ T cell populations were analysed via flow cytometry. The double antibody sandwich method was used to evaluate the expression of IL-2, IL-4, IL-12 and IFN-␥ in the sera collected from immunised BALB/c mice, per the manufacturer’s protocol (Jiamay Biotech, Beijing, China). A 96-well microplate was coated with the specific capture antibody and incubated overnight. On the second day, the plate was washed and blocked with 5% BSA, and sera samples were added, followed by incubation at room temperature for 2 h and subsequent application of an HRP-labelled detection antibody. Finally, the substrate TMB and an H2 SO4 stop solution were applied for colour development, and the optical density values were read at A450 . 2.5. Statistical analysis Statistical analysis was performed using SPSS 14.0 software for analysis of variance (ANOVA) and Duncan’s multiple range test. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Expression of the recombinant plasmids pMV261-TgCyP and pMV361-TgCyP in BCG The coding region of TgCyP was amplified via RT-PCR and cloned into both a shuttle expression vector, pMV261, and an integration expression vector, pMV361. The constructed plasmids were designated pMV261-TgCyP and pMV361-TgCyP and were verified by PCR
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Fig. 1. Western blotting assay of the expression of recombinant TgCyP protein in rBCG. Anti-T. gondii tachyzoite polyclonal antibody and HRP goat anti-mouse IgG were used to detect the specific expression of target proteins. N: negative control; 1: rBCGpMV361-TgCyP; 2: rBCGpMV261-TgCyP.
and sequencing. Positive clones were selected based on kanamycin resistance, and the selected rBCG bacteria were stimulated at 45 ◦ C to induce the expression of the target proteins. Western blotting showed that these recombinant plasmids were successfully expressed in vitro and were highly immunogenic (Fig. 1). 3.2. Humoral immune responses induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice Specific antibody responses against T. gondii tachyzoites were detected in all of the rBCG-vaccinated BALB/c mice through ELISA. Gradually increased IgG levels were observed in all of the rBCG- and BCG-vaccinated groups following each immunisation. From 2 to 6 weeks, all of the groups immunised with rBCG via the oral or i.v. route showed significantly higher IgG levels than the PBS groups (P < 0.001). Although the IgG level induced by BCG was elevated, it was still significantly lower than in the rBCG-vaccinated groups (P < 0.001; Fig. 2). 3.3. Cellular immune response induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice Splenocytes were harvested from mice and separated using lymphocyte separation medium. Flow cytometry analysis showed that the population of CD4+ T lymphocytes was significantly expanded in the rBCGpMV261-TgCyP orally vaccinated and the rBCGpMV261-TgCyP and rBCGpMV361-TgCyP i.v.-vaccinated groups. Furthermore, the percentage of CD8+ T lymphocytes increased gradually following rBCG immunisation. Thus, the i.v.
Fig. 2. Humoral immune responses induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP vaccines. BALB/c mice were immunized via either i.v. or oral route with rBCG vaccines. Serum were collected from 12 randomly mice of each group in 0 week, 2 weeks, 4 weeks and 6 weeks and measured for specific anti-T. gondii TLA antibodies by indirect ELISA. Data are represents by the mean OD ± S.E. (n = 12). Statistically significant differences are marked with the asterisk (compared with PBS group, * P < 0.05, **P < 0.01, ***P < 0.001).
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Table 1 Percentages of CD4+ and CD8* T cells in splenocytes from immunized BALB/c mice. Groups rBCGpMV261-TgCyPi.v. rBCGpMV261-TgCyP oral rBCGpMV361-TgCyPi.v. rBCGpMV361-TgCyP oral BCG j.v. PBS i.v.
CD4+ (%) 35.2 29.1 32.3 28.7 26.5 22.7
± ± ± ± ± ±
3.4. Protective efficacy of rBCGpMV261-TgCyP and rBCGpMV361-TgCyP in BALB/c mice
CD8*+ (%) *
0.28 0.25* 0.28* 0.16 0.18 0.21
18.32 15.60 17.1 15.4 15.8 14.5
± ± ± ± ± ±
0.16 0.14 0.12 0.15 0.12 0.19
Note: Data are represented by the Mean ± S.E. (n = 6). * Groups marked with asterisk are significantly different from the PBS control group (* P < 0.05).
route was more effective than the oral route in inducing a T. gondiispecific immune response (Table 1). Additionally, the levels of Th1-type IFN-␥, IL-2 and IL-12 and Th2-type IL-4 in vaccinated BALB/c mice were evaluated using a double antibody sandwich ELISA method. The ELISA results indicated higher levels of IFN-␥, IL-2 and IL-12 in the rBCGvaccinated groups, while the PBS and BCG groups showed only slight alterations in the levels of these cytokines (Fig. 3). Interestingly, intravenous injection induced the highest levels of IFN-␥, IL-2 and IL-12 observed in BALB/c mice, even compared to oral vaccination, and there was a significant difference detected compared to the control groups (P < 0.001). There was no difference between the experimental groups and control groups regarding the production of Th2-type IL-4 (P > 0.05). Furthermore, rapid production of IFN␥ early in the immune response, prior to the production IL-2 and IL-12, was observed in the rBCG groups.
Two weeks after the last vaccination, six mice were selected randomly from each group and challenged intraperitoneally with 103 highly virulent T. gondii RH strain tachyzoites. Overall, the rBCGvaccinated groups exhibited a higher protective efficacy than the control groups. The survival rate of the rBCGpMV361-TgCyP i.v.vaccinated group was 17% after 14 days, while the mice in all other groups expired prior to this time point (Fig. 4). Nevertheless, the rBCG-vaccinated group showed a prolonged survival time compared to the control groups. Thus, the i.v. route was superior to the oral route, as similar results were observed in terms of anti-T. gondii antibody production and the cellular response. Overall, the rBCGpMV261-TgCyP and rBCGpMV361-TgCyP vaccines limited T. gondii infection in BALB/c mice. 4. Discussion The present study was performed to determine the efficiency of recombinant BCG vaccines expressing the T. gondii cyclophilin gene in BALB/c mice. A significant and specific immune response was induced by the rBCG vaccines, administered either via i.v. or oral injection. Although the BCG vaccines were originally designed and tested through oral injection, the i.v. route is presumed to be the best way to administer rBCG vaccines. This study investigated the efficiency of i.v. injection of rBCG vaccines in mice. A protection efficiency of 17% was determined for the rBCGpMV361-TgCyP vaccine in BALB/c mice following challenge with T. gondii tachyzoites. As
Fig. 3. Cytokine production induced by rBCGpMV261-TgCyP and rBCGpMV361-TgCyP immunized mice. The serum of immunized mice was collected in 0 week, 2 weeks, 4 weeks and 6 weeks, and evaluated by ELISA for the Th1 and Th2 type cytokines production. (a) IFN-␥ production; (b) IL-2 production; (c) IL-4 production; (d) IL-12 production. Data are showed as mean ± S.D. (n = 6). Statistically significant differences are indicated by the asterisk (compared with PBS group, *P < 0.05, **P < 0.01, ***P < 0.001).
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Fig. 4. Survival rate of rBCGpMV261-TgCyP and rBCGpMV361-TgCyP immunized BALB/c mice after T. gondii challenge. Each mouse was given intraperitoneally with 103 T. gondii RH strain tachyzoites at 2 weeks after the last immunization (n = 6). Mice were monitored daily and survival times were recorded. The survival rate of rBCGpMV361TgCyP vaccinated mice can reach to 17%.
previously reported, the genetically stable integrative vector pMV361 was more efficient than the shuttle vector pMV-261 [29]. TgCyP has been indicated as a potential candidate gene for the development of an anti-T. gondii DNA vaccine [16]. This study was the first to express the T. gondii cyclophilin gene in exogenous attenuated Mycobacterium bovis. Overall, a significantly higher specific anti-T. gondii immune response was induced in rBCG vaccinated groups. Although the attained protection efficiency was low, BCG acted both as an adjuvant and a vehicle in eliminating T. gondii infection, and the TgCyP antigen played the dominant role in protection. BCG bacteria survive and replicate inside antigen-presenting cells (APCs), such as macrophages and dendritic cells. These APCs present BCG or heterologous antigens to initiate an immune response in mice [30]. rBCG induces both humoural and cellular immune responses against many heterologous antigens, including those from parasites, bacteria and viruses [31]. Similar to the results of a previous study, the TgCyP antigen was successfully expressed in BCG in the present work. In developing an rBCG vaccine, the main concern is the stability and expression efficiency of rBCG in vitro and in vivo. Multicopy plasmids can increase the expression level in an integrative BCG vector [31]. For some antigens, such as HIV-1 gp120, single copy integrative vectors are preferred, as high copy numbers may be lethal (e.g., 5 copies in pMV261) [18]. Because the pMV261 and pMV361 vectors utilise the highly transcript hsp60 promoter, they may be advantageous for rBCG vaccine development [18,32]. The pBMX vector, containing the hsp60 promoter, increases the expression of foreign antigens and elicits high antibody responses in BALB/c mice [33]. Similarly, a high level of specific anti-T. gondii IgG antibody production was observed in this study. Inoculation via the i.v. route results in the generation of specific antibodies and splenocyte proliferation in mice [32,34]. Mice immunised with rBCG via the oral route exhibit strong cellular and IgA responses to -galactosidase [35]. Administration through both the i.v. and oral routes in the present study induced a strong immune response against the BCG-expressed TgCyP antigen. Stronger anti-T. gondii immune responses were produced in the rBCG groups, with the greatest antibodies release being detected in the rBCGpMV361-TgCyP i.v. group. Administration via the i.v. route induced more antibody production compared to the oral route. I.v. immunisation also elicits a greater specific response than subcutaneous or intranasal inoculation against Schistosoma mansoni (S. mansoni) using Sm28GST [34]. This result may confirm the speculation that foreign antigens initiate different immune responses depending on the mode of immunisation [34]. Oral inoculation also significantly elevated antibody levels and cellular responses in
BALB/c mice. Although the immune responses to oral BCG vaccines are controversial, they are the best way for appropriate antigens to be taken up by the human gut [36–40]. As BCG induces shortterm resistance to S. mansoni, specific antibody responses should be stimulated by BCG, such as the increased antibody response observed in this study [41]. Recombinant proteins expressed by rBCG elicit more robust antibody responses than BCG antigen alone, which may persist much longer in mice [18]. In the present study, antibody production gradually increased in the rBCG groups, and a significant difference was observed even following the first inoculation, confirming that a single immunisation induces a strong immune response [18]. Likewise, specific immune responses were shown to be induced by T. gondii ROP2 expressed in BCG [23]. At longer times post-inoculation, the detected IgG levels were much higher, suggesting that rBCG induces a more efficient boost in foreign antigens and a stronger immune response. Green fluorescent protein expressed from the vector pMV261 is clearly visible inside human macrophages [42]. In general, cytosolic antigens are processed through the MHC class I pathway to induce CD8+ T cell production [43,44]. After BCG bacteria are killed, the released antigens are delivered to the phagolysosome and processed via the MHC class II pathway, predominantly stimulating CD4+ T cells [45]. The significantly increased production of CD4+ T cells observed in rBCG-vaccinated groups supports this hypothesis. The greater number of CD4+ T cells produced confirmed their dominant role in the rBCG-induced cellular immune response, while the increase in CD8+ T cells may have resulted from the initial cytotoxicity of BCG. CD4+ and CD8+ T cells are involved in the protection elicited by an rBCG-expressing membrane-associated protein [46]. rBCG inoculation enhances the Th1 response, as indicated by IFN-␥ levels, while suppressing the production of Th2-type IL-10 and IL-4 [47,48]. Th2-type IL-4, which is mainly involved in allergic reactions and asthma, cannot modulate BCG stimulation [49]. IL-12 is involved in the differentiation of naive T cells into Th1 cells and acts together with IL-18 to induce cell-mediated immunity following infection with microbial products such as lipopolysaccharide [50]. The secretion of IL-2 and IL-18 by the BCG strain enhances long-lasting Th1 phenotype-associated cellular immunity in mice [51–54]. IL-2 and IFN-␥ may modify the immune responses to BCG antigens [51,52,55]. Significantly high IFN-␥ levels have been observed in association with rBCG vaccines expressing the L. chagasi LCR1 antigen, S. mansoni Sm14 antigen and Leptospira interrogans LipL32 antigen [21,56,57]. In the present study, the levels of Th1-type IFN-␥, IL-2 and IL-12 were highly elevated following the administration of rBCG or BCG, while rBCG did not alter the
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production of Th2-type IL-4, suggesting that the Th1 response is the dominant cellular response to the BCG-expressed TgCyP antigen. Mice vaccinated with rBCG expressing membrane-associated proteins are better protected from challenge with Borrelia burgdorferi, Listeria or pneumococcal infection than mice receiving vaccines expressing cytosolic proteins [46,58,59]. Immunisation with rBCG expressing the C-terminal fragment of MSP-1 protects mice from Plasmodium yoelii challenge [60]. In the present study, we observed prolonged survival times in BALB/c mice vaccinated with rBCG. I.v. administration of the rBCGpMV361-TgCyP vaccine resulted in a protection efficiency of 17%. Higher humoural or cellular immune responses can be induced by linking a specific targeting sequence to a foreign antigen, such as the Mycobacterium tuberculosis 19 kDa lipoprotein, PspA signal sequence or mycobacterial ␣ antigen, compared to expression in the cytoplasm [39,46,59,61]. Thus, TgCyP delivered via BCG and expressed in the cytoplasm may confer limited protection against T. gondii challenge. We demonstrated that a BCG-expressing TgCyP vaccine was relatively effective in protecting BALB/c mice against T. gondii infection. Many factors, such as the promoter, site of expression, route of administration, dose administered and animal model involved, are of concern for rBCG vaccine development and for building the most effective BCG expression system for certain antigens. These considerations will prompt further studies investigating potential vaccine candidate antigens. Conflict of interest The authors have no competing interests. Acknowledgements This work was supported by a grant from the Special Fund for Agro-scientific Research in the Public Interest in China (no. 20130304). References [1] McCabe R, Remington JS. Toxoplasmosis: the time has come. The New England Journal of Medicine 1988;318:313–5. [2] Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clinical Microbiology and Infection: The Official Publication of The European Society of Clinical Microbiology and Infectious Diseases 2002;8:634–40. [3] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965–76. [4] Dubey JP, Jones JL. Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology 2008;38:1257–78. [5] Innes EA, Vermeulen AN. Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology 2006;133:S145–68. [6] Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clinical Microbiology Reviews 1998;11:267–99. [7] Cleary MD, Singh U, Blader IJ, Brewer JL, Boothroyd JC. Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryotic Cell 2002;1:329–40. [8] Radke JR, Behnke MS, Mackey AJ, Radke JB, Roos DS, White MW. The transcriptome of Toxoplasma gondii. BMC Biology 2005;3:26. [9] Quan JH, Chu JQ, Ismail HA, Zhou W, Jo EK, Cha GH, et al. Induction of protective immune responses by a multiantigenic DNA vaccine encoding GRA7 and ROP1 of Toxoplasma gondii. Clinical and Vaccine Immunology: CVI 2012;19:666–74. [10] Hiszczynska-Sawicka E, Li H, Xu JB, Holec-Gasior L, Kur J, Sedcole R, et al. Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154. Veterinary Parasitology 2011;183:72–8. [11] Mevelec MN, Ducournau C, Bassuny Ismael A, Olivier M, Seche E, Lebrun M, et al. Mic1-3 knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep. Veterinary Research 2010;41:49. [12] Ibrahim HM, Bannai H, Xuan X, Nishikawa Y. Toxoplasma gondii cyclophilin 18mediated production of nitric oxide induces Bradyzoite conversion in a CCR5dependent manner. Infection and Immunity 2009;77:3686–95. [13] Aliberti J, Valenzuela JG, Carruthers VB, Hieny S, Andersen J, Charest H, et al. Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells. Nature Immunology 2003;4:485–90.
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