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Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection

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Hai-Long Wang a,∗ , Min Pang b , Li-Tian Yin c , Jian-Hong Zhang a , Xiao-Li Meng a , Bao-Feng Yu d , Rui -Guo d , Ji-Zhong Bai e , Guo-Ping Zheng d,f , Guo-Rong Yin a,∗ a

Research Institute of Medical Parasitology, Shanxi Medical University, Taiyuan, Shanxi 030001, China Department of Respiratory, The First Affiliated Hospital, Shanxi Medical University, Taiyuan, Shanxi 030001, China c Department of Physiology, Key Laboratory of Cellular Physiology Co-constructed by Province and Ministry of Education, Shanxi Medical University, Taiyuan, China d Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, China e Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Private bag 92-019, Auckland 1142, New Zealand f Centre for Transplantation and Renal Research, the University of Sydney at Westmead Millennium Institute, Sydney, NSW 2145 Australia b

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Article history: Received 7 November 2013 Received in revised form 30 April 2014 Accepted 1 May 2014 Available online xxx

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Keywords: Toxoplasmosis Receptor for activated C kinase 1 Recombinant protein Intranasal immunisation Mucosal immune response Secretory IgA

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1. Introduction

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Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-␥ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-␥, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis. © 2014 Published by Elsevier B.V.

Toxoplasmosis is a worldwide distributed food borne zoonosis caused by the protozoan parasite Toxoplasma gondii, which infects the nucleated cells of warm blooded vertebrates, including humans (Dubey and Su, 2009). Usually, T. gondii infection is asymptomatic in immunocompetent humans, but it could be problematic in congenitally infected and immunocompromised individuals, such as AIDS patients and organ transplant recipients (Elsheikha, 2008; Innes,

∗ Corresponding authors at: Research Institute of Medical Parasitology, Shanxi Medical University, No. 56 Xinjian South Road, Taiyuan, Shanxi 030001, PR China. Tel.: +86 351 4135772. E-mail addresses: [email protected] (H.-L. Wang), [email protected] (G.-R. Yin).

2010; Weiss and Dubey, 2009). Toxoplasmosis in animals can also be a serious threat to public health and it can cause considerable economic losses of farm animals (Dubey et al., 2005). To prevent T. gondii infection, vaccination represents an optimal strategy (Kur et al., 2009). In the last two decades, many live, attenuated, subunit and DNA-based vaccines against toxoplasmosis have been studied, and a degree of progress has been achieved. For example, T. gondii surface antigen 1 (SAG1) and dense granule antigen 2 (GRA2) have been explored as vaccine candidates to protect against toxoplasmosis (Angus et al., 2000; Golkar et al., 2007), but only limited success against chronic and acute infection has been achieved to date (Dziadek and Brzostek, 2012). Therefore, further studies are necessary to develop appropriate vaccine antigens against toxoplasmosis. The receptor for activated C kinase 1 (RACK1) is a multifaceted scaffolding protein and serves as an integrative point for diverse

http://dx.doi.org/10.1016/j.actatropica.2014.05.001 0001-706X/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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signal transduction pathways. It links the Focal Adhesion Kinase (FAK) to the phosphodiesterase isoform PDE4D5 to control cell polarity (Serrels et al., 2011). It also interacts with the cytoplasmic tails of several receptors, including the Insulin-like Growth Factor Receptor I (IGF-IR), ␤-integrin receptor, the common beta-chain of the IL-5/IL-3/GM-CSF receptor (Geijsen et al., 1999) and modulates cell cycle, cell adhesion, cell spreading and cell migration (Cox et al., 2003; Mamidipudi et al., 2007). The RACK1 homologue for the parasite Trypanosoma brucei (TRACK) has a role in the cell cycle to ensure the mid-stage progression through cytokinesis in T. brucei procyclic forms (Rothberg et al., 2006). In Leishmania major, the RACK1 ortholog (LACK) is one of the major antigens which elicit a protective T cell response of the leishmaniasis subject (Julia et al., 1996; Mougneau et al., 1995). A RACK1 homologue expressed in the malaria parasite Plasmodium falciparum throughout its all asexual stages may also regulate the life cycle of malaria parasite (Madeira et al., 2003). RACK1 in T. gondii (TgRACK1) localises to the parasite cytoplasm and nucleus, and interacts with the ␤COP subunit of coatomer protein complex I involved in protein secretion and parasite invasion (Moran et al., 2007; Smith et al., 2007). TgRACK1 was later identified from soluble tachyzoite antigens by a rabbit anti-T. gondii serum and proteomic analyses, and was proposed to be a potential vaccine candidate against toxoplasmosis (Ma et al., 2009). T. gondii is an intracellular protozoan parasite that usually infects hosts through an oral route. The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. To evaluate the immune responses and protective efficacy of TgRACK1 against T. gondii, we cloned the TgRACK1 gene, prepared its recombinant protein, and investigated the effectiveness of the recombinant protein as a mucosal vaccine against T. gondii infection. 2. Materials and methods 2.1. Animals and parasites

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The mice used in this study were 6-week-old female BALB/c mice acquired from the Institute of Laboratory Animals, Chinese Academy of Medical Science, Beijing, China. All mice were maintained under standard conditions and provided with rodent feed and water ad libitum. Animal experiments were carried out in strict accordance to the Guidelines for Animal Experiments issued by the Ethics Committee of Shanxi Medical University under the licenses of 20110320-1. T. gondii tachyzoites (RH strain) were obtained from the Centre for Health Sciences of Peking University (Beijing, China) and maintained by serial intraperitoneal passaging in BALB/c mice.

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2.2. Expression and purification of recombinant TgRACK1

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was digested with EcoRI and NotI, and cloned into the vector pGEX-6P-1 (Merck Biosciences, Germany) using T4 DNA ligase (Transgen, China). The resulting pGEX-6P-1-TgRACK1 plasmid was transformed into E. coli BL21 (DE3) host cells (Transgen, China), and recombinant protein expression was induced with 0.1 mM IPTG at 20 ◦ C overnight. Cultures were harvested by centrifugation, cell pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 1 mM PMSF, 1 mM DTT) and homogenised by sonication on ice. The lysate was then centrifuged to separate the cell debris from the supernatant which was subject to 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting assay to verify the expression of rTgRACK1A by using anti-GST and anti-T. gondii polyclonal antibodies (Ma et al., 2009). The levels of rTgRACK1 expression and its purity were evaluated by SDS-PAGE and Coomassie blue R-250 staining. rTgRACK1 protein was purified using a self-packaged GSTaffinity column (2 ml glutathione Sepharose 6B; CoWin Biotechnology, China). Contaminant proteins were removed with a wash buffer (lysis buffer plus 200 mM NaCl) at 4 ◦ C. The fusion protein was then digested on column with PreScission protease (GE Healthcare, USA) at 4 ◦ C overnight, and rTgRACK1 protein without the GST tag at its N-terminus was eluted with the lysis buffer. Following centrifugation with an Ultrafree 10,000 molecular weight cut-off filter unit (Millipore, USA), the protein eluent was further purified using a Superdex-75 (Pharmacia, USA) column, and the purified protein was analysed by SDS-PAGE and Coomassie blue R250 staining. After endotoxin removal by using a ToxinEraserTM Endotoxin Removal Kit, the level of endotoxin remains in the final protein preparations was determined by using a Chromogenic End-point Endotoxin Assay Kit (Chinese Horseshoe Crab Reagent Manufactory, Xiamen, China) to be less than 0.1 EU/ml. The purified protein was then quantified by the BCA method, filtered throughout a 0.2 ␮m-pore membrane, and stored at −70 ◦ C until use. 2.3. Western blotting The products of rTgRACK1 expressed in E. coli were boiled in the gel loading buffer at 98 ◦ C for 5 min and were centrifuged at 12,000 × g for 10 min at room temperature (RT). The supernatants were separated on a 12% SDS-PAGE gel and transferred to a PVDF membrane (GE Healthcare, USA). The membrane was blocked in 5% (w/v) skim milk for 1 h at RT and then incubated with an antiGST antibody (1:1000) or rabbit anti-T. gondii serum (1:200) at 4 ◦ C overnight. After washes, the membrane was incubated with a HRPconjugated secondary anti-mouse or anti-rabbit antibody for 1 h at RT and developed with the ECL Western blotting analysis reagents (Engreen, China). 2.4. rTgRACK1 immunisation and samples collection

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Total RNA was extracted from tachyzoites of T. gondii using Trizol reagent according to the manufacturer’s instructions (Invitrogen, USA). First strand cDNA was synthesised using the EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, China). Primers for amplification of the open reading frame (ORF) of TgRACK1 gene were designed according to the gene sequence of TgRACK1 from T. gondii RH strain (GenBank accession No: AY547291). The forward primer was 5 -ACGGAATTCATGTCGGGTGAATCTCCCCTC-3 , and the reverse primer was 5 -AAGGAAAAAAGCGGCCGCTTACGCGGTCACTTGCTCTGAA-3 , which containing an EcoRI and NotI restriction site (underlined), respectively. The PCR conditions were as follows: 94 ◦ C for 5 min; 94 ◦ C for 30 s, 56 ◦ C for 30 s, 72 ◦ C for 1 min, 30 cycles; and 72 ◦ C for 10 min. For recombinant protein expression in Escherichia coli, the cDNA fragment of TgRACK1, which was confirmed via DNA sequencing,

Fifty 6-week-old female BALB/c mice were randomly divided into five groups (10 per group) and were intranasally immunised with 15, 25, 35 or 45 ␮g of rTgRACK1 dissolved in 20 ␮l sterile phosphate-buffered saline (PBS). Control mice received PBS alone. The mice were caught and their heads were hold in an upward position to expose their nostrils fully. To extenuate the distress of the mice, each dose of rTgRACK1 was instilled into the two nostrils alternatively (10 ␮l/nostril) within a period of 3 min per mouse by using a micropipettor. The same protocol of nasal inoculation was performed on days 0, 14, and 21. Two weeks after the final inoculation (on day 35), the mice were deprived of food and water for 8 h to deplete the contents in the intestines for intestinal wash collections. The mice were anesthetised with sodium pentobarbital (1.5%, 0.1 ml/20 g weight, intraperitoneal injection), and blood samples from the mice in each group were collected by retro-orbital plexus puncture. The sera were separated, stored at

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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−20 ◦ C, and then analysed for the presence of specific antibodies. Spleens were collected under aseptic conditions for lymphocyte proliferation assays, and the supernatants of isolated spleen cells were used for cytokine assays. Nasal washes were collected as described previously (Cisney et al., 2012; Matsuoka et al., 2009). By holding the mouse vertically, 0.1 ml of sterile PBS was carefully pipetted into one nostril and rinses were collected from the other nostril. A total of 0.6 ml of sterile PBS was flushed, and the rinse was transferred into a fresh tube. The intestinal washes were collected from the small intestine of the mice with 3.0 ml of PBS using previously described protocols (Goldoni et al., 2011). To collect the vesical washes, the bladder was exposed and rinsed gently with sterile PBS through the urethra by using a micropipette. The bladder was flushed six times with 0.1 ml PBS per flush, and the PBS was sucked back into the micropipette and re-injected into the bladder for a total of three cycles before final withdrawal. All the pooled mucosal washes collected at a total volume of 0.6 ml were centrifuged at 3,000 rpm for 10 min at 4 ◦ C to remove any tissue, faecal matter or cellular debris, and were stored at −20 ◦ C until assayed.

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containing only cells with medium. All assays were performed in triplicate. 2.7. Cytokine assays Cytokines were measured according to previously described method (Fachado et al., 2003). Splenocytes isolated from the immunised mice were cultured at 1.5 × 106 cells/well in the presence or absence of rTgRACK1 as described for the lymphocyte proliferation assay. Cell-free supernatants were harvested and assayed for interleukin-2 (IL-2) and IL-4 activities at 24 h, for IL-10 activity at 72 h, and for interferon-gamma (IFN-␥) activity at 96 h. The concentrations of IL-2, IL-4, IL-10 and IFN-␥ were evaluated by using a commercial ELISA kit (PeproTech, USA), according to the manufacturer’s instructions. The levels of IFN-␥ and IL-2 in intestinal washes were evaluated using the same protocols, and all assays were performed in triplicate. Cytokine concentrations were determined by reference to the standard curves constructed with known amounts of mouse recombinant IL-2, IL-4, IL-10 or IFN-␥. The detection limits of the assays were 16 pg/ml for IL-2 and IL-4, 47 pg/ml for IL-10, and 23 pg/ml for IFN-␥.

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2.5. Determination of anti-RACK1 antibodies Enzyme-linked immunosorbent assays (ELISAs) were used to detect antigen-specific secretory IgA (sIgA), IgG, IgG1 and IgG2a antibodies in serum samples which were taken two weeks after the last immunisation. In addition, antigen-specific sIgA in the nasal, intestinal and vesical washes was also measured using ELISA. Briefly, 96 flat-bottom wells of microtiter plates were coated overnight at 4 ◦ C with 1 ␮g rTgRACK1 in 100 ␮l sodium carbonate buffer (pH 9.2). The plates were washed with PBS containing 0.05% Tween 20 (PBST), pH 7.4. PBS containing 5% fetal calf serum (FCS) was used for blocking non-specific binding sites for 2 h at 37 ◦ C. After washing three times (5 min each wash) with PBST, individual sera (100 ␮l/well) diluted in 1% BSA-PBST (1:200 for IgG, 1:50 for IgA, IgG1 and IgG2a) and nasal, intestinal or bladder washes (100 ␮l/well) were applied to the wells and incubated for 1 h at 37 ◦ C. Following the wells being washed, bound antibodies were detected by adding 50 ␮l horseradish peroxidase-conjugated goat anti-mouse IgA, IgG, IgG1 or IgG2a (ProteinTech Group, Inc., USA) at 1:2000 dilutions. After washings with PBST, immune complexes were revealed by incubating with orthophenylene diamine (Sigma, USA) and 0.15% H2 O2 for 30 min, and the enzyme reaction was terminated with the addition of 50 ␮l of 1 M H2 SO4 . The optical density (OD) was measured with an ELISA plate reader (Epoch Multi-Volume Spectrophotometer System, Biotek, USA) at 492 nm. All samples were analysed in triplicate.

2.6. Lymphocyte proliferation assay Under aseptic conditions, spleens were collected in Hank’s balanced salt mixture (Solarbio, China) from the animals 2 weeks after the last immunisation. They were minced using a pair of scissors and passed through a 300 mesh sieve (41.6 ␮m) to obtain a homogeneous cell suspension. The lymphocytes were isolated in lymphocyte isolation solution (Biyuntian, China), and were cultured in 96-well plates at 2 × 105 cells/well in RPMI-1640 containing 10% FCS. The cells were stimulated with either rTgRACK1 (10 ␮g/ml), concanavalin A (Con A: 5 ␮g/ml, Sigma) or medium alone (negative control) for 72 h at 37 ◦ C in 5% CO2 . The number of cells was determined with the cell counting Kit-8 (Dojindo, Japan), and results were expressed as the stimulation index (SI). The latter was calculated as the ratio of the average OD450 value of wells containing antigen-stimulated cells to the average OD450 value of wells

2.8. Challenge experiment To determine the efficacy of rTgRACK1 against T. gondii infection, mice immunised with 35 ␮g of rTgRACK1 were selected for chronic and lethal challenge, according to the immune response data obtained above for rTgRACK1 at different doses. The BALB/c mice immunised with rTgRACK1 and PBS control mice (30 mice/group, 10 mice for chronic infection and 20 mice for lethal assay) were orally challenged with either 1 × 104 tachyzoites for the chronic infection or 4 × 104 tachyzoites for an acute infection on the fifteenth day after the last immunisation. On the 31st day, the numbers of tachyzoite in the murine brains and livers were determined by real-time PCR assay to assess the chronic infection. For the acute challenge, the time to death of the challenged mice was monitored and recorded at 8 am, 2 pm and 8 pm daily, and the survival was assessed for 30 days after the parasite challenge. 2.9. DNA extraction and real-time PCR assay Genomic DNA from the purified parasites, the liver and brain samples (100 mg each) were extracted by using a UniversalGen DNA Kit (CWBIO, China), according to the manufacturer’s instructions. The forward and reverse primer sequences of SAG1 gene were 5 -CTGATGTCGTTCTTGCGATGTGGC-3 and 5 -GTG AAGTGGTTCTCCGTCGGTGT-3 , respectively. PCR was performed using the Applied Biosystems® Real-Time PCR Instruments and SYBR Green fluorescence detection. Each reaction mixture contained 12.5 ␮l of UltraSYBR Mixture (CWBIO), 0.4 ␮l of each primer (20 ␮M), 1 ␮l of DNA template and 10.7 ␮l of sterile distilled water. Sterile water was used as negative control, and a DNA extracted from 500 tachyzoites of the T. gondii RH strain was used as positive control. All reactions were in triplicate and incubated for 1 min at 95 ◦ C followed by 40 cycles of 5 s at 95 ◦ C, 15 s at 60 ◦ C and 10 s at 72 ◦ C (Yu et al., 2013). The number of parasites in the samples was calculated from the qPCR threshold cycle (Ct) value according to a standard curve (linear curve slope: −2.9756, Y intercept: 34.4193) obtained with DNA samples from a range of serial 10-fold dilutions (5 × 100 to 5 × 107 /ml) of RH strain tachyzoites under the same conditions. The tachyzoite loads in the liver and brain samples were presented as mean value of the quantity of tachyzoites estimated in per gram tissues.

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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Fig. 1. SDS-PAGE and Western blotting analyse of rTgRACK1. (A) Expression of rTgRACK1 in E. coli upon IPTG (0.1 mM) induction at 20 ◦ C was detected by 12% SDS-PAGE and Coomassie blue staining at molecular weight approximately 63 kDa. (B and C) rTgRACK1 was detected by Western blotting analyses using an anti-GST antibody (B) or a rabbit anti-T. gondii polyclonal antibody (C). (D) Purified rTgRACK1 protein was visualised via 12% SDS-PAGE and staining with Coomassie blue; the purity of rTgRACK1 was greater than 95%. M: Standard protein marker; lane 1: Protein expression without IPTG induction; lane 2: Total protein expression induced by IPTG; lane 3: Proteins from the lysate pellet of the induced-expression system; lane 4: Proteins from the lysate supernatant of the induced-expression system. Asterisk (*) represents non-specific protein bands.

2.10. Statistical analysis

3.2. rTgRACK1 vaccination evokes mucosal immune responses

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The differences in the levels of cytokine production, antibody production, lymphocyte proliferation and tachyzoite loads were determined by one-way ANOVA. Results were considered to be statistically significant at P < 0.05 for the immunised mice when compared to PBS treatment controls. The survival time was analysed and compared between the immunised and control groups using the Kaplan–Meier method.

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To investigate the mucosal immune response of rTgRACK1 vaccination in mice, the levels of specific sIgA in the nasal, intestinal and vesical washes were determined by ELISA analysis two weeks after the last immunisation. The titers of rTgPACK1-specific sIgA antibody in the mucosal washes were increased following nasal immunisation (Fig. 2). The sIgA antibody titers in the nasal washes of the 15, 25, 35 or 45 ␮g rTgRACK1-treated groups were significantly higher than those of the PBS control (P < 0.05 for the 15 ␮g rTgRACK1 group, and P < 0.01 for the other groups), with the highest titer of sIgA antibody detected in the 35 ␮g rTgRACK group (Fig. 2A). sIgA levels from the intestinal and vesical washes were higher in mice that were nasally immunised with 25, 35, 45 ␮g rTgRACK1 compared with those from the PBS control and 15 ␮g rTgRACK1 groups, and 35 ␮g rTgRACK1 could also elicit the highest sIgA levels in intestinal and vesical washes (P < 0.01) (Fig. 2B and C). No significant difference was found between the mucosal washes of the 35 and 45 ␮g rTgRACK1-treated groups. When the levels of IFN-␥ and IL-2 in the intestinal washes were analysed by ELISA, enhanced production of both IFN-␥ and IL-2 were detected in the 25 ␮g rTgRACK1 group (P < 0.05) and reached higher levels in the 35 and 45 ␮g rTgRACK1 groups (P < 0.01) when compared with PBS controls (Fig. 3). These results suggest that nasal

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The ORF of the TgRACK1 was cloned into the prokaryotic expression vector pGEX-6P-1 and was transformed into a BL21 (DE3) bacterial host. After induction with 0.1 mM IPTG at 20 ◦ C overnight, the GST-rTgRACK1 protein was successfully expressed in E. coli at a molecular weight of approximately 63 kDa (Fig. 1A). The fusion protein was detected by Western blotting analysis with anti-GST antibody (Fig. 1B) and rabbit anti-T. gondii polyclonal antibodies (Fig. 1C). The isolated rTgRACK1 protein is approximately 36 kDa after the GST Tag removed from its N-terminal. It was water soluble, and had a 95% purity by SDS-PAGE gel analysis (Fig. 1D).

Fig. 2. Intranasal rTgRACK1 vaccination induces specific sIgA production in mucosal washes. Groups of BALB/c mice were immunised with different doses of rTgRACK1 vaccine on days 0, 14 and 21. Two weeks after the last inoculation, samples of nasal, intestinal and vesicle washes were collected to determine the levels of sIgA by ELISA. Dose-dependent production of sIgA was detected in nasal washes (A), intestinal washes (B) and vesical washes (C) collected from mice that were nasally immunised with rTgRACK1 compared to those from the PBS controls. Results are expressed as means of OD492 ± SD (n = 10) from three independent experiments. * P < 0.05, ** P < 0.01 (vaccinated vs. controls).

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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Fig. 3. rTgRACK1 vaccination stimulates IFN-␥ and IL-2 release in intestinal washes. Mice were immunised with different doses of rTgRACK1 at 0, 14, 21 days, and the intestinal washes were collected 35 days later to detect the levels of IFN-␥ (A) and IL-2 (B). Increased levels of both IFN-␥ and IL-2 were observed in mice immunised with rTgRACK1 compared to PBS controls. Data represent the mean value ± SD (n = 10) from three experiments. * P < 0.05, ** P < 0.01 (vaccinated vs. controls).

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rTgRACK1 vaccination in mice provokes immune responses with increased productions of sIgA, IFN-␥ and IL-2 in their mucosal tracks of the nasal, bladder and intestine.

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3.3. rTgRACK1 vaccination evokes specific antibody productions

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To investigate whether mice immunised with different doses of rTgRACK1 displayed a humoral immune response, sera were collected from the mice and rTgRACK1-specific antibodies were analysed by ELISA. Significantly increased levels of total IgG antibody were observed in the 25, 35 and 45 ␮g rTgRACK1-treated groups compared to the control group (P < 0.05) (Fig. 4A). The maximum IgG antibody response was detected in the 35 ␮g rTgRACK1-treated group, and no significant difference was found between the 35 ␮g and 45 ␮g groups (P > 0.05). Similarly, when the levels of IgG1 and IgG2a humoral isotypes were evaluated by ELISA in the sera collected from the immunised BALB/c mice, a mixed IgG1/IgG2a response with predominant IgG2a production was detected in the mice immunised with 25, 35 and 45 ␮g rTgRACK1 compared to PBS controls (Fig. 4B). Significant response of IgA antibodies were also detected in the 25, 35 and 45 ␮g groups compared to the control group (P < 0.05) (Fig. 4C). Taken together, these data show that rTgRACK1 immunisation via a nasal route also triggers a humoral immune response with increased production of serum IgGs and IgA.

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3.4. rTgRACK1 vaccination promotes certain cytokine production

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Production of Th1-type (IFN-␥ and IL-2) and Th2-type cytokines (IL-4 and IL-10) represents an important means of cell-mediated

immunity (Dimier-Poisson et al., 2006; Yamamura et al., 1992). When these parameters in the rTgRACK1-immunised mice were evaluated, by measuring the amounts of cytokines released in the supernatants of spleen cell cultures obtained from the mice, it was found that rTgRACK1 immunisation stimulated dose-dependent production of cytokines IFN-␥, IL-2 and IL-4 but not IL-10 in the isolated spleen cells (Fig. 5). Significant productions of IFN-␥, IL-2 and IL-4 were all evident at 25 ␮g rTgRACK1 treatment and reached a maximum level at 35 ␮g rTgRACK1 (Fig. 5A–C). No significant changes in production of IL-10 were seen in all the mice immunised with different dosage of rTgRACK1 (P > 0.05) (Fig. 5D). Therefore, these data indicate that rTgRACK1-induced cellular immune response displays a mixed Th1- and Th2-type response which is predominately Th1 oriented. 3.5. rTgRACK1 vaccination stimulates lymphocyte proliferation To further determine the cell-mediated immune responses of rTgRACK1 vaccination, splenocytes were isolated from the mice immunised with PBS or different doses of rTgRACK1 and lymphocyte proliferation assay was performed upon further rTgRACK1 stimulation in vitro. As shown in Fig. 6, rTgRACK1 immunisation promoted a dose-dependent proliferation of the lymphocytes when they were challenged with rTgRACK1 at 10 ␮g/ml for 72 h in culture. Significant increase in the numbers of lymphocyte (expressed as the stimulation index, SI) was seen at 25 ␮g rTgRACK1 immunisation and reached a maximum level at 35 ␮g rTgRACK1 (P < 0.01, Fig. 6), similar to those cells isolated from the same mice and treated with 5 ␮g/ml concanavalin A (Con A) as a positive control (data not shown). In line with the above results of cytokines

Fig. 4. rTgRACK1 vaccination promotes specific anti-rTgRACK1 humoral responses in mice. Two weeks after the final immunization, blood samples were obtained and determined by ELISA using rTgRACK1 as the specific bound target for the levels of total IgG (A), IgG1 and IgG2a isotypes (B) and IgA (C) in the sera from mice vaccinated with rTgRACK1 or from control animals. Results are expressed as means of the OD492 ± SD (n = 10) from three experiments. * P < 0.05, ** P < 0.01 (vaccinated vs. controls).

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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Fig. 5. rTgRACK1 vaccination promotes cytokine releases from isolated splenocytes of the immunised-mice. Splenocytes were harvested from the mice 2 weeks after the last immunisation and were cultured in the presence or absence of rTgRACK1 (10 ␮g/ml). Supernatants were assessed for the production of IFN-␥ (A) at 96 h, IL-2 (B) and IL-4 (C) at 24 h, and IL-10 (D) at 72 h by ELISA. Data represent the mean value ± SD (n = 10) from three experiments. * P < 0.05, ** P < 0.01 (vaccinated vs. controls).

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production in stimulated spleen cells, these data suggest that rTgRACK1 vaccination elicits systemic immune responses in mice. 3.6. rTgRACK1 vaccination protects mice against T. gondii infection To evaluate whether rTgRACK1 vaccination protects against T. gondii, we employed the mouse model of tachyzoite infection

Fig. 6. rTgRACK1 vaccination stimulates lymphocyte proliferations. Splenocytes harvested from the mice 2 weeks after the last immunisation were cultured in vitro with or without rTgRACK1 (10 ␮g/ml) for 72 h. The number of cells was then determined using the cell counting Kit-8 assay. Results are expressed as the mean stimulation index (SI) ± SD (n = 10) from three experiments. ** P < 0.01 (vaccinated vs. controls).

via an oral route as previously reported by us and others (Wang et al., 2013; Yin et al., 2007; Yin et al., 2013). During a chronic infection, control mice and mice immunised with 35 ␮g rTgRACK1 were orally infected with 1 × 104 tachyzoites on the fifteenth day after the last immunisation and were monitored daily for further 15 days after the challenge. The numbers of tachyzoite in the mouse brain and liver tissues were determined by real-time PCR assay. As shown in Fig. 7A, significant reductions of tachyzoite were observed in both the liver and brain tissues of rTgRACK1-immunised mice in comparison with those of PBS controls, respectively. The numbers of tachyzoites in the brain tissues were 1.12 ± 0.17 × 106 /g tissue for the rTgRACK1-immunised mice, and 2.09 ± 0.32 × 106 /g tissue for the PBS controls, representing a 57.09% reduction with rTgRACK1 vaccination (P < 0.01). Similarly, the numbers of tachyzoites in the liver tissues were (2.61 ± 0.28) × 106 /g tissue for the rTgRACK1-immunised mice, and (5.63 ± 0.74) × 106 /g tissue for the PBS controls, showing a 53.64% reduction with rTgRACK1 vaccination (P < 0.01). These findings suggest that rTgRACK1-elicited immunity reduces the burdens of tachyzoite infection in its host tissues. When the protective effect of rTgRACK1 vaccination against an acute lethal T. gondii infection was monitored in mice challenged orally with 4 × 104 tachyzoites, it was found that the death time of mice immunised with 35 ␮g rTgRACK1 was between 8 and 21 days after the challenge, while all of the mice in the PBS control group died from 6 to 10 days after the challenge (Fig. 7B). rTgRACK1-immunised mice significantly increased their survival rate (approximately 45%) at the end of the challenge compared with those of the PBS control group (P < 0.01). These data indicate that rTgRACK1 immunisation at least partially protects mice against acute infection with T. gondii RH strain.

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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Fig. 7. Intranasal rTgRACK1 vaccination protects mice against T. gondii infection by oral route. (A) Tachyzoite burdens in brain and liver tissues collected from rTgRACK1immunised or control mice (10/group) which were orally challenged with 1 × 104 tachyzoites of the T. gondii RH strain for 30 days. Data are presented as the average quantity of tachyzoites per gram of tissue from three independent experiments. ** P < 0.01 (vaccinated vs. controls). (B) Survival rate of rTgRACK1-immunised or control mice (20/group) during 30 days after oral challenge with 4 × 104 tachyzoites of the T. gondii RH strain. All mice immunised with PBS controls died within 10 days, and the mice immunised with rTgRACK1 died between 8 and 21 days. At the end of the challenge, nine mice in vaccinated group (45%) were alive.

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4. Discussion T. gondii receptor for activated C kinase 1 (TgRACK1) is involved in a signalling event that precedes protein secretion and parasite invasion via interaction with ␤COP (Moran et al., 2007; Smith et al., 2007). The RACK1 homologue in L. major (LACK) elicits a protective T cell response against the leishmaniasis subject (Julia et al., 1996; Mougneau et al., 1995). Moreover, TgRACK1 has been identified from soluble tachyzoite antigens and it reacts with a rabbit anti-T. gondii serum, suggesting that this protein could serve as a novel vaccine candidate (Ma et al., 2009). In this study, we purified rTgRACK1 from an E. coli expression system and verified it as a protective immunogen for intranasal immunisation in BALB/c mice. Nasal rTgRACK1 vaccination in mice promoted the productions of sIgA, IFN-␥ and IL-2 in mucosal tracks, and the productions of total IgG, IgG1 and in particular IgG2a in sera. It stimulated lymphocyte proliferation and release of cytokines IFN-␥, IL-2, IL-4 but not IL-10 in isolated spleen cells from the immunised mice. Reduced burdens of tachyzoite in host tissues and increased host survival rate were observed when the rTgRACK1-immunised mice were challenged with T. gondii during chronic and acute lethal infections, respectively. These results suggest that rTgRACK1 is an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis. Vaccination is the most effective biomedical strategy for preventing infections. As over 90% of infectious diseases are initiated by pathogens that traverse mucosal surfaces, stimulation of the mucosal immunity is the optimal approach to control such infections. This stimulation is best achieved through vaccination in the body’s mucosal openings (Holmgren and Czerkinsky, 2005) such as nasal administration which is a practical, non-invasive method of inoculation (Bumann et al., 2010; Xue et al., 2010). T. gondii can infect the gut mucosa by direct invasion of epithelial cells in the small intestine (Dubey et al., 1997). Therefore, the intestinal mucosa can act as the first defence line where epithelial cells may respond directly to a T. gondii infection and initiate early local mucosal immune responses. In line with these studies, here we demonstrated that intranasal vaccination of rTgRACK1 significantly increased the level of sIgA in the nasal, intestinal and vesicle washes and also IFN-␥, IL-2 in the intestinal washes of the mice vaccinated. It is well established that host defences at mucosal surfaces include the secretion of IgA, cytokines and chemokines. sIgA is regarded as the major defence player due to the fact that pathogen-specific sIgA can inactivate the pathogen and contribute to pathogen elimination. sIgA is also generally more

cross-reactive against pathogen variants than IgG and other classes of immunoglobulins. IFN-␥ and IL-2 are essential for the induction of antigen-specific sIgA responses (Finkelman et al., 1990). Thus, increased levels of both IFN-␥ and IL-2 were also detected in the intestinal washes of rTgRACK1-immunised mice. IFN-␥ is thought to be a marker for protective immunity against T. gondii (LaRosa et al., 2008). It controls both acute and chronic infection with T. gondii (Aliberti, 2005), restricts the growth of T. gondii in the acute phase, and prevents reactivation of parasites from dormant cysts at a later phase (Jones et al., 1986). IL-2 can augment the IFN-␥ production in the secondary response to an intracellular pathogen and strengthen the roles of IFN-␥ in control T. gondii (Sa et al., 2013). IFN-␥ and IL-2 can also modulate sIgA (Estes, 2010; Marinaro et al., 1997), control the infection with T. gondii at intestine mucosal sites (Aliberti, 2005), and participate in gut homeostasis and recruitment of immune cells during infections (Ju et al., 2009). Therefore, our data suggest that rTgRACK1 vaccination via nasal route can induce effective mucosal immune responses. Consistent with the above findings of rTgRACK1-stimulated mucosal releases of sIgA, IFN-␥ and IL-2, both humoral and cell-mediated immunities were evident in the immunised mice with the increased productions of antibodies including total IgG, IgG1 and IgG2a isotypes, and IgA from blood serum, and of cytokines IFN-␥, IL-2, IL-4 from their isolated spleen cells. When the levels of humoral isotypes IgG1 and IgG2a were evaluated, a mixed IgG1/IgG2a response with predominant IgG2a production (IgG2a/IgG1 ratio > 1) was revealed in the rTgRACK1- immunised mice. These data indicates that rTgRACK1 challenge predominantly activated the Th1-type immune response. This is supported by the finding of rTgRACK1-stimulated productions of IFN-␥, IL-2 and IL-4 but not IL-10. It is thought that IFN-␥ and IL-2 generally correlate with Th1-type immune responses, whereas IL-4 and IL-10 correlate with Th2-type responses (Fraternale et al., 2011; Jafarzadeh and Shokri, 2012). Being consistent with the production of cytokines, we also observed significant enhancement of lymphocyte proliferations by rTgRACK1 vaccination. Together, these data suggest that nasal rTgRACK1 vaccination in mice induces a humoral and cellular immune response, and that this response is Th1- and Th2-mixed but predominately Th1 oriented. There are three clonal lineages of T. gondii that differ in their abilities to induce cytokines and virulence. Type I is regarded as a virulent strain, while type II and type III are attenuated strains (Sibley and Boothroyd, 1992). Mice infected with type I parasites show widespread parasite dissemination and rapid death,

Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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and they are mostly used to assess the immunoprotective effect of novel antigens (Meng et al., 2012; Zhou et al., 2012). In this study, we employed the virulent RH strain of T. gondii admitted by an oral route to evaluate the protection effect of rTgPACK1 protein against T. gondii infection in mice. We observed significant reductions of tachyzoites in the liver and brain tissues of the rTgPACK1-immunised mice but not the control animals during a chronic infection. The vaccinated mice also displayed significant protection against lethal load of the tachyzoites, showing about 45% improved survival rate and prolonged life spans from 8 to 21 days. This suggests the effectiveness of rTgRACK1 immunisation compared with the use of other recombinant proteins, such as recombinant T. gondii actin depolymerising factor or nucleoside triphosphate hydrolase-II in similar studies where all immunised mice died within 9 or 14 days (Huang et al., 2012; Tan et al., 2011). Thus, TgRACK1 immunisation is effective to reduce the burdens of tachyzoite infection in its host tissues, and protects its host against T. gondii infection. T. gondii infections by oral acquisition of the apicomplexan protozoan include three infectious stages of the parasites: tachyzoites, bradyzoites, and sporozoites (Dubey, 1998). Although the latter two stages are the main components of natural T. gondii infections by oral taken of the parasite’s tissue cysts or oocysts, the rapidly dividing tachyzoites can disseminate the infection to virtually all organs and tissues of the host and even reach the fetus transplacentally. While bradyzoite-containing cysts are often used as oral infectious materials in studies (Awobode et al., 2013; Boyle et al., 2007; Gregg et al., 2013), an oral route of tachyzoite infection has approved to be effective in cats and mice (Dubey, 2005; Penarete-Vargas et al., 2010; Wang et al., 2013). A mouse model of tachyzoite infection via an oral route has also been previously established by us (Wang et al., 2013; Yin et al., 2007; Yin et al., 2013). In this model, 1 × 104 tachyzoites were used to infect mice by oral gavage. The infected mice showed rough coat, decrease in appetite, weakness/inability to obtain feed or water. Importantly, tachyzoites were detectable in all the organs and tissues of infected mice. In this study, significant higher numbers of tachyzoites were also evident in the liver and brain tissues of PBS control animals than those of the rTgRACK1-immunised mice (Fig. 7). These results suggest that tachyzoites can infect mice by an oral route, although some tachyzoites could be killed by gastric juice that needs to be further explored. Although our results of an oral challenge with tachyzoites appear to be less comparable with others via different routes, such as intraperitoneal tachyzoite injection (Qu et al., 2008; Zhao et al., 2013; Zheng et al., 2013), our model system provides an alternative and useful approach to evaluate the role of mucosal immune response for protecting against T. gondii infection. In summary, data obtained in this study suggest that intranasal immunisation with rTgRACK1 induces both mucosal and systemic immune responses, and is effective in providing protection against T. gondii infection. Further studies on the enhancement of the vaccine potential of the recombinant antigen, such as through combination in vaccine formulation with effective adjuvants (e.g. the cholera toxin) that promote mucosal sIgA and systemic IgG productions (van Ginkel et al., 2005), could lead to the development of novel mucosal therapies against toxoplasmosis.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81071374), the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No. 20111010); the Technique Innovation of Shanxi Medical University (No. 01201103) and the 331 Early Career Researcher

Grant of Shanxi Medical University (No. 201202). This work was supported by the Biology Postdoctoral Mobile Research Station of Shanxi Medical University.

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Please cite this article in press as: Wang, H.-L., et al., Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.05.001

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Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection.

Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resista...
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