Immunization with rEmCRT provides protection against Echinococcus  multilocularis infection in BALB/c mice

doi:10.21203/rs.3.rs-1740725/v1

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Immunization with rEmCRT provides protection against Echinococcus multilocularis infection in BALB/c mice

https://doi.org/10.21203/rs.3.rs-1740725/v1

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Background

Alveolar echinococcosis (AE) is a severe parasitic zoonosis, caused by the larval stage of Echinococcus multilocularis. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Echinococcus infection.

Methods

First of all, we predicted the physical and chemical properties and protein structure characteristics of E. multilocularis calreticulin (EmCRT), and then constructed the recombinant plasmid pET28a-EmCRT. Secondly, the constructed recombinant plasmid was transformed into E. coli BL21 and induced to produce recombinant EmCRT (rEmCRT). Then the antigen specificity and the calcium-binding property of rEmCRT was detected. Thirdly, the expression level and distribution of Emcrt gene in PSCs and vesicles were analysed. And the protective immunity induced by rEmCRT immunized mice and the corresponding mechanismwas investigated in the end.

Results

We identified that EmCRT, a ubiquitous protein with Ca²⁺-binding ability, elicited protective immunity against E. multilocularis infection. EmCRT was found to be expressed on the surface of E. multilocularis larvae as well as in the secreted products of metacestode vesicles and protoscoleces (PSCs). Recombinant EmCRT formulated with Freund’s adjuvant (FA) elicited both IgG1 and IgG2a antibodies, with a higher IgG1/IgG2a ratio indicating a predominant Th2 response in vaccinated mice, and stimulated mouse lymphocytes from the spleen to produce high levels of Th1 (IFN-γ, IL-2) and Th2 (IL-4, 5, 10) cytokines. The protection was around 43.7% after challenged with E. multilocularis PSCs.

Conclusions

Our results suggest that EmCRT is an immunodominant protein secreted by E. multilocularis larvae and contributes to the induction of partial protective immunity in vaccinated mice against Echinococcus infection. Therefore, EmCRT could be a novel and potential candidate vaccine against AE.

Echinococcus multilocularis

alveolar echinococcosis

calreticulin

vaccine

protective immunity

Alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by the larval stage of Echinococcus multilocularis, which is much rarer but far more severe than Cystic echinococcosis (CE) resulting from the growth of E. granulosus metacestodes [1, 2]. The primary organ affected is the liver and it causes cyst-like lesions in organs of infected individual in 99% of cases. In addition, infection can also extend locally or metastasize relying on lymphatics or blood vessels to distant sites, such as the spleen and brain [3]. More than 1 million people are affected with echinococcosis at any one time in the world. Many of these people will be experiencing severe clinical syndromes which are life-threatening, and the 10-year mortality of untreated AE patients is greater than 90% after diagnosis [4]. There is clear evidence that this parasite can easily spread from endemic to non-endemic areas. Despite the preventive measures against Echinococcus infections conducted for a long time, few countries have been able to substantially reduce or eradicate AE.

In most cases, up to 75% of the infected people may remain free symptom for more than 10 years. Currently, the treatment of AE is mainly through surgery which is always accompanied by chemotherapy [4, 5]. However, surgery cannot completely remove the parasite tissue in most cases, because of the diffuse and infiltrative feature of the metacestode tissue. Treatment with drugs mainly relies on benzimidazoles, but they can only restrain the growth and dispersion of metacestodes, they can't eliminate the parasite [5]. They show limited efficiency against AE and cause severe side effects.

Calreticulin (CRT) is a well-conserved and ubiquitous protein with Ca²⁺-binding ability. It exists in the endoplasmic reticulum of all kinds of cells, except that erythrocytes [6]. CRT is a multifunctional protein and has been identified in certain parasites [7–9]. In addition, studies have found that CRT of some parasitic helminths, e.g. Taenia solium, hookworm and Schistosoma mansoni, could induce partial protection after immunization [10–13]. In this study, we identified and characterized a CRT homologue, EmCRT, in E. multilocularis. Significant protection against E. multilocularis infection was elicited by immunization with recombinant EmCRT in mice. These findings are of interest in the evaluation of this protein as a vaccine candidate against AE.

Parasite and animals

E. multilocularis used in this study was obtained from Hulunbeier Pasture of Inner Mongolia of China [14]. Protoscoleces (PSCs) and vesicles were isolated from in vivo cultivated parasite material and cultured in vitro as described by literature [15–18].

The 6-8-week old female BALB/c mice (weighting 18–21 g) were purchased from the Xiamen University Laboratory Animals Center (XMULAC). The mice were raised and housed at the XMULAC with free access to food and water [19, 20].

Nucleic acid isolation, cloning, expression, and purification of recombinant EmCRT (rEmCRT)

Total RNA was extracted from the vesicles and the cDNA was synthesised according to the manufacturer’s instructions. The DNA encoding the full-length EmCRT without the signal peptide (1–18 amino acids) was amplified by PCR with the following primers which were designed from nucleotide sequences of EmCRT (UniProtKB accession No. A0A068YA41): the forward primer was 5′-ATGGGTCGCGGATCCATGGAAGTTTACTTC-3′, and the reverse primer was 5′-GTGGTGGTGCTCGAGCTACAATTCATCCTT-3′. The PCR product was digested with BamH I and Xho I and subcloned into the expression vector pET-28a (kept by our group) and sequenced. The rEmCRT was expressed in E. coli BL21 (DE3) under induction of 0.5 mM IPTG at 25℃ for 4 h. The soluble rEmCRT with His-tags at N-terminus was purified by Ni-affinity chromatography (Beyotime, China) according to the manufacturer’s instructions. The purity of rEmCRT was analyzed by SDS-PAGE and confirmed by Western blot with the anti-His tag antibody (Cell Signaling Technology, USA) [19–21]. The protein concentration was determined using BCA Protein Assay Kit (Beyotime). Endotoxin (LPS) was removed by ToxinEraserTM Endotoxin Removal Kit (GenScript, China) and resulting in < 0.1 endotoxin units/mL, as measured by the ToxinsensorTM Chromogrnic LAL Endotoxin Assay Kit (GenScript) according to the manufacturer’s protocol [22].

Calcium-binding staining

The calcium-binding property of rEmCRT was assessed by staining with Stains-all (Sigma, USA), a cationic carbo-cyanine dye that stains Ca²⁺-binding proteins blue and all other proteins red [23–25]. In the assay, bovine serum albumin (BSA) was used as the control. In addition, the recombinant protein of EmBmi-1 (EmuJ_001132200), a molecular being investigated in another project, was used as an E. multilocularis irrelevant control.

Expression of native EmCRT in E.multilocularis metacestode larvae

To analyze the transcription of the Emcrt gene in different developmental stages of E. multilocularis, total RNA was extracted from E. multilocularis PSCs and vesicles with the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The cDNA templates were reverse-transcribed from the same amount of total mRNA of PSCs and vesicles with the Reverse Transcription Kit (ACCURATE BIOLOGY, China). The constitutively expressed E. multilocularis gene (Emelp) was used as an internal control. Primers for detection of the Emcrt gene were designed as follows: 5′-TACACGCTCATCATCCGACC-3′ (forward) and 5′-TTCCGGTTGTTTGGCATTCG-3′ (reverse). Primers for Emelp were 5′- CAGGATCTCTTCGATCAAGTG-3′(forward)and 5′-GACCATACTTGGCAACACAG-3′ (reverse). Real-time quantitative PCR (RT-qPCR) was performed to determine gene transcription levels in PSCs and vesicles by using HieffTM qPCR SYBR® Green Master Mix (YEASEN, China) in the Roche LightCycler 96. Fluorescence was acquired after each extension phase. After amplification, a melting curve analysis was performed to assess the specificity of the product. Standard curves were generated with the dilution of cDNA starting at a 1:5 [26, 27]. The results of the threshold cycle (Ct) were calculated using 2^−ΔCt method after being normalized by Emelp, and the fold-change of the Emcrt gene expression level in E. multilocularis PSCs was calculated relative to that in vesicles.

For detection of EmCRT protein expression, anti-serum was generated by immunizing mice with rEmCRT as described [22]. PSCs and vesicles were isolated from Kunming mice and in vitro cultivated as described before [15–18]. Crude somatic extracts of PSCs and vesicles were obtained by mechanical rupture and centrifugation. At the same time, we gathered the vesicles fluid protein. In addition, we collected some PSCs and vesicles and cultured them in RPMI 1640 (Shanghai Basalmedia Technologies Co., LTD, China) for 48 h at 37°C with 5% CO₂. The culture supernatants containing excretory-secretory (ES) products were concentrated by centrifugation and buffer exchanged into phosphate-buffered saline (PBS) [22]. Afterwards all protein samples were separated by SDS-PAGE with 10% polyacrylamide gel, then transferred to PVDF membranes. Detection was performed with the anti-rEmCRT serum and a secondary anti-mouse IgG antibody conjugated with HRP (1:5,000 dilution; BOSTER Biological Technology co. ltd, china).

Immunofluorescence assay (IFA)

For localization of EmCRT in the metacestode vesicles, immunofluorescence assay was performed as described previously using the anti-rEmCRT serum as the primary antibody [19].

For immunofluorescence localization of EmCRT in the PSCs, the primary antibody was incubated for five days and the secondary antibody (Alexa 488-conjugated antibody, Life Technologies, USA) for two days. For all immunofluorescence experiments, DNA was counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Fluorescence images were taken using a Nikon 80i fluorescence microscope. Contrast and light adjusting and merge of pictures were done by using Nikon image software supplied by the microscope manufacturer or Adobe Photoshop 8.0 software [28–30].

Immunization schedule and challenge infection

BALB/c mice were randomly divided into three groups with 13 animals each. The mice in the first group were each vaccinated subcutaneously with 25 µg of rEmCRT emulsified with complete Freund’s adjuvant (FA) [31, 32]. After 2 weeks, BALB/c mice were immunized with incomplete FA to boost immunization. The second and third groups were inoculated with FA emulsified with PBS alone, or with PBS only, as controls using the same immunization method as the first group [33, 34].

ELISA measurement of the antibody response

Mice sera from inner canthus were collected one week before each vaccination and measured for rEmCRT-specific IgG by using an indirect enzyme-linked immunosorbence assay (ELISA). Briefly, flat-bottom, 96-well cell culture plate (Thermo Fisher, USA) were coated overnight at 4°C with 100 µL rEmCRT at a concentration of 1.0 µg/mL in bicarbonate buffer (pH 9.6). After three washes with PBS + 0.05% Tween-20 (PBST), the 96-well cell culture plate were blocked with blocking buffer for 1 h at 37°C. After another three washes with PBST, the microplates were probed with serial dilutions of immune sera for 1 h at 37°C. The plates were then washed and incubated with HRP-conjugated goat anti-mouse IgG for 1 h at 37°C. After the final wash, the substrate 3, 3’, 5, 5’-tetramethylbenzidine (TMB, Beyotime) was added to each well, and the reactions were stopped with stop solution (Beyotime). Quantification of the reactions was determined by measuring the absorbance at 450 nm with an ELISA reader [33, 35].

The IgG subclasses were measured with ELISA kit according to the manufacturer’s instructions [36, 37].

Spleen cell culture and cytokine analysis

Two weeks after the last immunization, 5 mice from each group were sacrificed and their spleens were removed under aseptic conditions. After being counted, each mouse’s cells at a density of 5×10⁵/100 µL were cultured in a 96-well cell culture plate. Each sample was inoculated with 9 wells, three wells were stimulated with 10 µg/mL rEmCRT at 37℃ for 72 h in the presence of 5% CO₂ followed by the addition of 10 µL/well CCK-8 (Cell Counting Kit-8, MCE, USA) solutions. The plates were then incubated for 4 h in the dark. Splenocytes stimulated with 5 µg/mL concanavalin A (Con A, Sigma) served as positive controls in other three wells, the last three wells were handled with medium only as negative control. Three groups were analyzed using the same processing method as the first group. Then the absorbance of the samples at 450 nm was measured [35, 36].

Real time qPCR was performed to assess the expression of IL-2 and IFN-γ (Th1 type cytokines), and IL-4, IL-10, IL-5 (Th2 type cytokines) mRNAs in spleens of mice that were vaccinated but not infected. Total RNA extraction from the spleen and cDNA synthesis were conducted as described earlier. We designed primers for GAPDH, IL-2, IL-10, IL-5, IFN-γ and IL-4. Relative mRNA expression was obtained with a Light Cycler 96 instrument (Roche, Applied Science, Germany), using a SYBR® Green Master Mix (YEASEN). A PCR reaction (20 µL) contained 2 µL cDNA, DEPC H₂O 7.2 µL, 0.4 µL of each primer and 2×HieffTM qPCR SYBR® Green Master Mix (YEASEN) 10 µL. The PCR cycling conditions were: initial denaturation at 95℃ for 5 min followed by 40 cycles, each consisting of denaturation at 95℃ for 10 s, annealing at 56–63℃ (depending on the primer sequence) for 20 s, and extension at 72℃ for 20 s. For IFN-γ and IL-4 the annealing temperature was 60℃, for IL-2 and IL-5 was 56℃, and for IL-10 and GAPDH was 61℃. After amplification, the results of the threshold cycle (Ct) were calculated using 2^−ΔCt method after being normalized by GAPDH, the fold-change of the group of rEmCRT emulsified with FA was calculated relative to that in FA emulsified with PBS alone, or with PBS only [38–40].

Evaluation of alveolar echinococcus weight

Two weeks after the final boost, the remaining 8 mice in each group were challenged 2000 PSCs by intraperitoneal injection. All mice were sacrificed 14 weeks after infection, and the total parasite material was collected to measure the parasite weight [41].

Statistical analysis

The results of western blot were scanned using the analytic software ImageJ. The data were expressed as the means ± standard error. p < 0.05 was regarded as statistically significant. Statistical analysis was performed using the Prism 7.0 software (GraphPad Prism software, USA). Analysis was carried out using the two-tailed Mann–Whitney test.

Cloning of Emcrt gene

DNA encoding for EmCRT without the signal peptide was amplified from E. multilocularis vesicles cDNA by PCR using gene-specific primers designed on Emcrt sequence. The amplified 1,137 bp DNA fragment was cloned into E. coli expression vector pET-28a.

The BLASTP analyses of the conserved P-terminal portion of EmCRT according to protein databases with that of other species are shown in Fig. 1a, it revealed that its closest relatives were Echinococcus granulosus calreticulin. In addition, by using the EmCRT sequence as a query in BLASTP analyses, we identified calreticulin orthologs in Taenia solium (TsM_000094800), Ancylostoma duodenale (Ancduo_03806), and Schistosoma mansoni (Smp_030370), which encoded proteins with 89, 58, and 48% amino acid sequence identity to EmCRT, respectively.

Phylogenetic analysis of the calreticulin family also showed that EmCRT is closely related to the calreticulin homologue in Echinococcus granulosus (Fig. 1b). The dendrogram basically reflects the evolutionary relationships of the organisms involved. We generated a model for EmCRT tertiary structure by SWISS MODEL, with three major domains (N-terminal, internal P and C-terminal) are present in our deduced EmCRT sequence (Fig. 1c).

Recombinant EmCRT (rEmCRT) expression and identification

To detect the expression of EmCRT in E. multilocularis, we constructed a recombinant plasmid encoding EmCRT without the signal peptide in vector pET-28a, leading to translational fusions of the E. multilocularis protein with an N-terminal 6×His tag. The His-tagged rEmCRT protein could be recognized by the anti-His antibody, exhibiting a molecular weight of approximately 58 kDa (Fig. 2a) which differed from the predicted molecular weight of 46 kDa including the His tags. The anomalous migration in SDS-PAGE may be due to the high negative charge of EmCRT (pI=4.7) or other structural features (variable glycosylation after protein synthesis) [42-45].

Western blot results revealed that rEmCRT was strongly recognized by rEmCRT-immunized mice sera as well as E. multilocularis-infected mice sera. No reaction was observed with normal sera (Fig. 2b). The results indicate that EmCRT is a highly immunogenic antigen and induces a strong antibody response in hosts during the infection.

We also found that rEmCRT could be stained in blue with Stains-all, while other proteins without calcium-binding activity were stained in red (Fig. 2c), suggesting that rEmCRT is a calcium binding protein.

Em CRT expression in E. multilocularis metacestode larvae

RT-qPCR was performed to observe the transcriptional level of Emcrt gene in E. multilocularis PSCs and vesicles. As shown in Fig. 3a, we found that the expression level of Emcrt in PSCs was significantly higher (4.1-fold) compared with that in the vesicles (**p < 0.01).

The protein expression level of native EmCRT was determined by Western blot with the anti-rEmCRT serum. As shown in Fig. 3b, one single 58-kDa protein band was clearly identified in the extracts from both PSCs and vesicles. The protein load was much higher in the PSCs sample, consistent with the results of RT-qPCR. Furthermore, EmCRT expression was also detected in the vesicle fluid and the culture supernatants of the PSCs and vesicles.

The IFA results showed that EmCRT was highly presented in the germinal layer of vesicles (Fig. 4a). EmCRT was also actively expressed in the PSCs, and some strong signals were detected on the surface of PSCs, mainly in the zones with a greater nuclear density, particularly around the suckers. In contrast, little reactivity was detected in PSCs and vesicles when probed with normal mouse sera (Fig. 4b). Taken together, these results demonstrate that EmCRT is constitutively present throughout E. multilocularis larval stages and acts an excretory-secretory product of the parasite.

Additional files shows these datas with more details in Additional file 1 to 2.

Immunogenicity of r Em CRT

The serum samples were gathered and the antibody titers against rEmCRT were measured using ELISA. The vaccination of mice with rEmCRT formulated with FA induced significantly higher levels of rEmCRT-specific IgG than the control group and the highest IgG titer reached 1:512,000 after the third immunization (Fig. 5a).

The number of splenic lymphocytes from the mice immunized with rEmCRT emulsified with FA was significantly increased when the cells were stimulated with rEmCRT or ConA. Control groups had little effect (p > 0.05). The data suggest that the proliferation of splenocytes from immunized mice can be induced in vitro in response to stimulation with rEmCRT (Fig. 5b).

Furthermore, the levels of IgG subclass antibody were measured to assess the efficacy of rEmCRT inducing the response of different IgG subclass in vivo. The results showed that the levels of IgG1 and IgG2a were significantly elevated after the last 2 immunizations, with IgG1 predominating (Fig. 5c). It favors a Th2-biased immune response.

In order to evaluate changes in the cytokines mRNA expression at the systemic level in response to rEmCRT + FA immunization, spleen samples were obtained two week after the last boost. Our results showed that the levels of typical Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-5, IL-10) were all increased significantly in the mice vaccinated with rEmCRT compared with the FA emulsified with PBS alone and PBS groups, indicating that rEmCRT vaccination induced mixed Th1 and Th2 responses. In addition, the Th2 cytokines IL-4 and IL-5 increased more significantly, suggesting that rEmCRT may favor to induce a Th2-biased immune response (Fig. 6), consistent with the result of IgG subclass antibody levels.

An additional file shows this data with more details in Additional file 3.

Partially protective immunity elicited by r Em CRT

Vaccine efficacy of EmCRT was observed by the alveolar echinococcus weight in immunized mice. The result showed that the immunized group exhibited a 43.7% mean reduction in the parasite weight after 14 weeks of challenge relative to the control group (p < 0.05). There was no significant difference in the alveolar echinococcus weight between PBS with the FA and PBS alone control groups. These results indicate that rEmCRT enables the induction of partial protective immunity against E. multilocularis infection in mice (Table 1).

Table 1

Alveolar echinococcus weight in mice.
Groups	Alveolar echinococcus weight
Groups	range	average value
rEmCRT + FA	0.5253–1.5416	0.8539125	0.6483275	43.7%*
PBS + FA	0.5566–1.8407	1.1720375	0.3302025
PBS	0.8466–2.6281	1.50224
*Statistically significant.
Alveolar echinococcus weight in mice each group after challenge with 2000 protoscoleces. Alveolar echinococcus weight are presented as the mean ± S.D. The asterisks indicate statistically significant differences in alveolar echinococcus weight compared to the control group (p < 0.05).

An additional file shows this data with more details in Additional file 4.

The metacestode stage of E. multilocularis is characterized by its tumor-like proliferation, causing the life-threatening AE in humans [1–3]. At present, there is lack of an effective prevention and a reliable early diagnostic method for E. multilocularis infection. Therefore, the development of effective vaccines for AE are needed. Proteins are often the first choice for most researchers when evaluating candidates for vaccine antigens as they play crucial roles in parasite growth and metabolism, and are relatively easy to identify and manufacture [46].

CRT is a well-conserved Ca²⁺-binding protein with multiple biofunctions. After searching the genome database, we found two genes encoding the CRT homologues in E. multilocularis (locus EmuJ_000653100 and EmuJ_000653200). We then refer to EmuJ_000653100 as Emcrt and EmuJ_000653200 as Emcrt2 to distinguish them. In this study, our work mainly focused on Emcrt due to its higher homology with other parasite CRT. Sequence alignment shows that EmCRT shares 50–90% identity with CRTs from other parasites, including T. solium, hookworm and S. mansoni. All of these CRTs have been identified in the secreted form or as expressed on the parasite surface such as Trypanosoma carassii, Necator americanus, Trichinella spiralis, and Brugia malayi [22, 47–49].

In our previous study, we utilized the bioinformatics online software to predict the secondary structure and T and B cell antigen epitopes of EmCRT. Our findings showed that EmCRT contains a typical signal peptide at the N-terminal of first 18 amino acids, and 6 joint epitopes of T and B cells [50]. The expression of EmCRT was detected in both the vesicles and PSCs of E. multilocularis at the mRNA and protein levels. Furthermore, native EmCRT was localized on the surface of the PSCs around suckers and the germinal layer of vesicles, and was also present in the excretory-secretory (ES) products of vesicles and PSCs, and cystic fluid of vesicles, suggesting its importance in the interaction between parasite and host and immunological accessibility if being developed as a vaccine.

The cellular immune and the humoral immune response could give full play to remove parasites when host meets the “correct” antigen, because they may relate to the mechanisms of protective immunity induced by recombinant protein immunization and play an important role in protective immunity for helminth infections [35, 51, 52]. In this study, BALB/c mice were immunized with rEmCRT formulated with FA and induced higher levels of rEmCRT-specific IgG and the highest IgG titer reached 1:512,000 after the last immunization. The levels of IgG subclass and cytokine for splenocytes were evaluated at the same time, the results indicated that rEmCRT vaccination induced mixed Th1 and Th2 responses in mice like other parasite CRTs and indicated a predominant Th2 response as far as results were concerned. The number of spleen cells from mice immunized with recombinant protein significantly increased when stimulated with rEmCRT or ConA. It is suggested that the recombinant protein vaccine can stimulate lymphocyte proliferation in vitro. On the whole, our data further indicated that EmCRT is a highly immunogenic antigen and induces a strong antibody response in hosts and contribute to observe the protective immunity in this study.

There is growing evidence supporting that parasitic CRT plays important roles in regulation of adaptive immune responses in diverse parasitic disease. For example, T. solium CRT (TsCRT) favors a Th2-biased immune response characterized by the induction of IgG1, IL-4, IL-10 and IL-5 [12–13], when reddish bigger parasites were used for challenge after vaccination of 9 month old hamsters with TsCRT, around 40% protection was induced; native hookworm CRT can induce low levels of serum IgE and moderate lung eosinophilia. Meanwhile, the worms in lungs of mice with immunized calreticulin had alleviated 43–49% which compared to non-vaccinated controls [11]. While S. mansoni CRT acts as a strong T-cell immunogen and is capable of inducing IL-4 synthesis [10, 53], indicating that CRT is able to induce a Th2 response and immune protective effect during helminth infections [54]. Based on these reasons, we have further explored the E. multilocularis calreticulin for its potential as a vaccine against E. multilocularis infection.

However, the alveolar echinococcus weight reduction induced by immunization with rEmCRT against E. multiocularis PSCs challenge in this study was not very high (alveolar echinococcus weight reduction is 43.7% compared PBS control), further research is needed to construct a new vaccine with EmCRT. We also have made a discussion according to the result. The reason largely results from the fact that the complex life cycle of parasites, diversity of antigens, another contributing factors are immune-evasion strategies and the modulatory effect of host responses [55]. In addition, the clinical symptoms of the alveolar echinococcus is relying on the location, the size of the cyst and the development stage [56].

This work demonstrates that EmCRT plays an important role in inducing partially protective immunity in the host and therefore could be considered a potential candidate for vaccine development against AE. But we also need to change the immune pathway and mode or the combine multiple effective vaccines to get deeper research for the vaccine of alveolar echinococcosis.

Alveolar echinococcosis

Echinococcus multilocularis

Cystic echinococcosis

CRT

Calreticulin

PSCs

Protoscoleces

XMULAC

Xiamen University Laboratory Animals Center

IPTG

Isopropyl-beta-D-thiogalactopyranoside

SDS-PAGE

sodium salt Polyacrylamide gel electrophoresis

BSA

bovine serum albumin

RT-qPCR

Real-time quantitative PCR

threshold cycle

RMPI

Roswell Park Memorial Institute

excretory-secretory

PBS

phosphate-buffered saline

DAPI

4',6-diamidino-2-phenylindole

ELISA

enzyme-linked immunosorbence assay

CFA

complete Freund’s adjuvant

HRP

Horseradish Peroxidase

TMB

3, 3’, 5, 5’-tetramethylbenzidine

CCK-8

Cell Counting Kit-8

ConA

concanavalin A

IL-2

Interleukin-2

IL-4

Interleukin-4

IL-5

Interleukin-5

IL-10

Interleukin-10

IFN-γ

Interferon-gamma.

Acknowledgements

We would like to Prof. Tang Chongti for kindly providing the parasite material. and Yaying Wu, Zheni Xu and Dr. C.C. Xie for mass spectrometry experiments and data anslysis.

Funding

This study was financially supported by grants from the National Natural Science Foundation of China (81860362) and Natural Science Foundation of Inner Mongolia Autonomous Region (2018BS08013).

Availability of data and materials

Data and material used in the studies are available.

Authors’ contributions

LZ, LC conceived and designed the study. LC, ZX, and ZC performed the experiments. LC, ZX, SX, ZX, YY, and JC analyzed the data. LC, ZC wrote the paper. LZ, YW, ZC, and LC revised the manuscript. All authors reviewed the manuscript.

Ethics approval and consent to participate

The protocol was in strict accord with good animal practice as defined by XMULAC. Animal experiments including in vivo propagation of parasite material in Kunming mice were approved by the Institutional Animal Care and Use Committee of Xiamen University, China (approval Number: 2013-0053) and in strict accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Laboratory of Pathogenic biology, School of Basic Medical Sciences and Forensic medicine, Baotou Medical College, Baotou, Inner Mongolia, China

²Parasitology Research Laboratory, School of Life Sciences, Xiamen University, Xiamen, Fujian, China

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No competing interests reported.

Additionalfile1.zip
Amino acid sequence analysis and predicted three-dimensional structure of EmCRT, characterization of EmCRT, expression of EmCRT in E. multilocularis metacestode larvae.
Additionalfile2.zip
Immunolocalization of EmCRT in E. multilocularis metacestode larvae.
Additionalfile3.zip
The antibody titers and IgG subclass of the serum samples and splenocyte proliferation assay with rEmCRT in vitro, cytokines mRNA expression after stimulation with rEmCRT in vitro.
Additionalfile4.xlsx
Alveolar echinococcus weight in mice.

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Immunization with rEmCRT provides protection against Echinococcus multilocularis infection in BALB/c mice

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Parasite and animals

Nucleic acid isolation, cloning, expression, and purification of recombinant EmCRT (rEmCRT)

Calcium-binding staining

Immunofluorescence assay (IFA)

Immunization schedule and challenge infection

ELISA measurement of the antibody response

Spleen cell culture and cytokine analysis

Evaluation of alveolar echinococcus weight

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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