Vaccination of mice with a recombinant novel cathepsin B inhibits Trichinella spiralis development, reduces the fecundity and worm burden

Background Trichinella spiralis is a major zoonotic tissue-dwelling nematode, which is a public health concern and a serious hazard to animal food safety. It is necessary to exploit an anti-Trichinella vaccine to interrupt the transmission of Trichinella infection among animals and from animals to humans. The purpose of the present study was to characterize the novel T. spiralis cathepsin B (TsCB) and to evaluate the immune protection elicited by immunization with recombinant TsCB (rTsCB). Methods The complete cDNA sequences of the TsCB gene were cloned, expressed and purified. The antigenicity of rTsCB was investigated by western blot analysis and ELISA. Transcription and expression of TsCB at various T. spiralis life-cycle stages were analyzed by RT-PCR and indirect immunofluorescent assay (IIFA). The mice were subcutaneously immunized with rTsCB, and serum level of TsCB-specific IgG (IgG1 and IgG2a) and IgE antibodies were assayed by ELISA. Immune protection elicited by vaccination with rTsCB was investigated. Results The TsCB was transcribed and expressed in four T. spiralis life-cycle stages (adult worm, AW; newborn larvae, NBL; muscle larvae, ML; and intestinal infective L1 larvae), it was primarily located in the cuticle and stichosome of the parasitic nematode. Vaccination of mice with rTsCB produced a prominent antibody response (high level of specific IgG and IgE) and immune protection, as demonstrated by a 52.81% AW burden reduction of intestines at six days post-infection (dpi) and a 50.90% ML burden reduction of muscles at 35 dpi after oral larva challenge. The TsCB-specific antibody response elicited by immunization with rTsCB also impeded intestinal worm growth and decreased the female fecundity. Conclusions TsCB might be considered as a novel potential molecular target to develop vaccines against T. spiralis infection.

and areas [3][4][5]. Infections with Trichinella spp. are not merely a public health concern but also a severe hazard to animal food safety [6,7]. It is difficult to eradicate Trichinella spp. infection in animals as preventive anti-Trichinella vaccines are not currently available. [8,9]. Screening and identification of Trichinella spp. invasion-related proteins is recommended to help identify novel candidate targets for a vaccine against Trichinella infection [10].
After being eaten, T. spiralis ML encapsulate in the skeletal muscles and are released from their capsules in the stomach, where they develop into intestinal infective L1 larvae (IIL1) within the intestines. The IIL1 larvae intrude into enteral epithelia and continue to grow into adult worms (AW) by molting four times [11,12]. Female adults give birth to newborn larvae (NBL), which pass into the bloodstream, penetrate into the skeletal muscles and encapsulate to accomplish the life-cycle [13]. The intestinal epithelial invasion by IIL1 larvae is the first infection, but the invasion mechanism is not clear. As intestinal epithelia are the preferential natural barrier against larval invasion, and the major site for host-T. spiralis interaction [14,15], identification of IIL1 invasive proteins will be valuable to understand invasion mechanisms of the parasite and develop vaccines against T. spiralis intestinal invasive worms [16,17].
Cathepsin B is one member of the cysteine protease family, which plays an important function in worm invading, migrating, molting and immune escape [18,19]. Cysteine proteases have been identified in excretion/ secretion (ES) products or somatic proteins of T. spiralis ML and AW [20,21]. When T. spiralis IIL1 larvae were inoculated onto an enteral epithelium cell monolayer, the IIL1 larvae penetrated the monolayer and expressed additional cysteine proteases which were found to be highly expressed at the IIL1 stage [22]. It might participate in IIL1 intrusion of the enteral epithelium during Trichinella infection [23][24][25].
In the present study, a novel cathepsin B gene of T. spiralis (TsCB, GenBank: XP_003379650.1) was obtained from the T. spiralis draft genome [26], cloned and expressed. The TsCB were characterized and the protective immunity triggered by rTsCB immunization were investigated in a mouse model.

Worm maintenance and experimental animals
Trichinella spiralis (ISS534) isolated from a domestic pig in central China was maintained in mice by serial passage in our laboratory [27]. Six-week-old female BALB/c mice were provided by the animal centre at Zhengzhou University.

Worm collection and antigen preparation
The ML were recovered by artificially digesting T. spiralis-infected mouse muscles at 35 days post-infection (dpi) [28,29]. The IIL1 were isolated from mouse intestine at 6 hpi [30], and the AW were collected from mouse intestine on days 3 and 6 after infection [31]. After washes with sterile PBS, the day 6 AW were cultured in RPMI-1640 medium (Gibco, Auckland, New Zealand) containing 10% fetal bovine serum (50 female worms/ml), and the newborn larvae (NBL) were recovered 24 h following culture [32]. The soluble proteins of ML, IIL1, AW and NBL, and the ML ES proteins were prepared as previously reported [33,34].

RT-PCR quantification of TsCB transcript levels
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) from worms of the different lifecycle stages (ML, IIL1, 3 days AW and NBL). TsCB transcript level at each stage was quantified by RT-PCR as reported [40]. Trichinella spiralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank: AF452239) was amplified as a housekeeping gene [41]. PBS was utilized as a negative control in all PCR amplification.

Immunization of mice and analysis of antibody responses to rTsCB
Sixty mice were divided into three groups of equal size (20 mice/group). Each mouse was subcutaneously vaccinated with 20 µg rTsCB emulsified with ISA 201 adjuvant (Seppic, Paris, France). Vaccination was repeated three times at 2-week intervals using the same dose of rTsCB and ISA 201. Control groups received only ISA 201 or PBS using the same vaccination procedure [44]. Individual serum samples were collected before vaccination and on weeks 2, 4, 6 and 8 after vaccination [45].

Indirect immunofluorescence assay (IIFA)
Expression and tissue localization of natural TsCB in the nematode were investigated using IIFA with anti-rTsCB serum [42,52]. Paraffin sections (3 µm thick) of the different worm life-cycle stages were used to examine TsCB expression and tissue localization in T. spiralis. Each section was blocked with 5% normal goat serum (Sangon, Shanghai, China), and probed at 37 °C for 1 h with three different sera (1:10; anti-rTsCB serum, mouse infection serum and pre-immune serum). Following three washes with PBS, the sections were stained at 37 °C for 1 h using FITC-conjugated anti-mouse IgG (1:100; Santa Cruz, USA). Sections were washed as previously reported and examined using fluorescent microscopy (Olympus, Tokyo, Japan) [49,53].

Challenge experiment
To investigate the immune protection offered by vaccination with rTsCB, all mice were infected orally with 300 T. spiralis ML at two weeks after the final boost. Intestinal adults were collected from 10 mice at 6 dpi [54], and muscle larvae at 35 dpi were obtained by artificially digesting the carcasses of the remaining 10 mice [55]. Immune protection was ascertained as worm reduction of enteral adults and larvae per gram (LPG) of skeletal muscles of immunized groups compared to those of the PBS control group [8,56,57].

Statistical analysis
All statistical analysis was conducted using SPSS for Windows, version 22.0 (SPSS Inc., Chicago, IL, USA). The values are presented as the mean ± standard deviation (SD). Difference among various groups was analyzed using a Student's t-test or one-way ANOVA. P < 0.05 was regarded as a level for statistical significance.

Bioinformatics analysis of TsCB sequence
Bioinformatics analyses revealed that the full-length TsCB sequence was 1071 bp, encoding a protein of 356 amino acids, with 40.23 kDa and 7.86 isoelectric point (pI). Analyses with Signal P 4.1 and TMHMM Server indicated that the signal peptide was located at 1-29 aa, TsCB had 7 α-helixes and 13 β-strands, and a transmembrane domain was located outside the cell membrane. Subcellular localization of TsCB was present in mitochondria (2%), periplasm (94.9%) and cytoplasm (6.7%), respectively. The homology comparison of TsCB sequences with those of other Trichinella species or genotypes are shown in Fig. 1. TsCB amino acid sequence had 98% identity with cathepsin B of T. nativa, T. murrelli, T6, T8, T. britovi and T. nelsoni, and 95% identity with T. pseudospiralis.
Phylogenetic analysis of TsCB with cathepsin B from other species is shown in Fig. 2a. The phylogenetic tree generated using the MP method verified a monophyletic group of the above-mentioned 7 species/gene types Fig. 1 Sequence alignment of TsCB with cathepsin B from Trichinella spp. and other species. Sequence alignment was conducted in Clustal X and shown using BOXSHADE. Black shade indicates that residues were the same as TsCB; conservative substitutions are marked in grey. The percentage identity with TsCB is shown within the genus Trichinella. Trichinella spiralis has a close evolutionary relationship with encapsulated and non-encapsulated Trichinella species, and is more closely related to nematode cathepsin B than that from other species.
The SMART analysis results revealed that there was a functional domain (between positions 102-351 aa) of peptidase_C1A. In a 3-dimensional model, TsCB had the catalytic active sites, which were composed of Gln124, Cys130, His300 and Asn320 residues, forming a pocketshaped functional domain carrying substrate binding sites (Fig. 2c).

RT-PCR analysis of TsCB transcription
Transcription of the TsCB mRNA was assayed by RT-PCR for the four parasite life-cycle stages and the GAPDH gene was used as an internal control. A TsCB transcript (984 bp) was detected in muscle larvae, IIL1, adults and NBL. Primers for GAPDH also generated the expected size (570 bp) at all stages (Fig. 3).

Western blot identification of rTsCB
The results of SDS-PAGE revealed that the BL21 bacteria carrying PQE-80L/TsCB expressed a 39.7 kDa fusion protein. After purification, the rTsCB protein exhibited a clear individual band (Fig. 4a). The molecular weight (MW, 39.7 kDa) of rTsCB was identical to its predicted size.
Western blotting results exhibited that rTsCB was recognized by anti-rTsCB antibodies, but not by infection serum and normal mouse serum (Fig. 4b). Using anti-rTsCB antibodies, native TsCB was detected in soluble proteins of muscle larvae, IIL1, 3-days adults and NBL, but not in muscle larva ES proteins (Fig. 4b, c), indicating  B (b, c). b The predicted 3-dimensional structure of TsCB contains 7 α-helixes (red) and 13 β-strand (blue). c Functional domain carrying catalytic reactive sites consisted of Gln124, Cys155, Asn305, and Gly328 residues, formed a functional domain. The TsCB active sites are highlighted in red that the TsCB is one somatic protein of this nematode, but not a secretory protein from muscle larvae.

Expression and localization of TsCB in the nematode life-cycle stages
The IIFA result revealed that the fluorescence staining was detected in the four life-cycle stages (ML, IL1, 3-days female adult and embryos) by anti-rTsCB antibodies. The fluorescence was distributed in the cuticle and stichosome of the nematode and in the embryos within female uterus (Fig. 5). Fluorescence staining with pre-immune serum was not observed.

Specific antibody response
To determine specific antibody responses to rTsCB, rTsCB-specific IgG, IgG1 as well IgG2a, and IgE in serum samples of vaccinated mice, responses were measured using an rTsCB-ELISA. The anti-rTsCB IgG titer was 1:10,000 after the third immunization (Fig. 6), indicating that the rTsCB was a strong immunogenic. The anti-rTsCB IgG level in vaccinated mice was prominently raised following the second immunization, whereas no mice vaccinated with ISA 201 or PBS exhibited any anti-rTsCB antibody responses (Fig. 7a). The IgG1 levels at 4, 6 and 8 weeks post-immunization were prominently higher than IgG2a (week 4: t (18) = 4.350, P < 0.0001; week 6: t (18) = 4.247, P < 0.0001; week 8: t (18) = 2.902, P = 0.009) (Fig. 7b, c), demonstrating that immunization with rTsCB elicited a Th2-predominant Th1/Th2 mixed immune response. Moreover, anti-rTsCB IgE was also determined, and the results showed that the specific IgE level was significantly elevated in mice immunized with rTsCB in comparison to the control groups (F (4, 45) = 568.102, P < 0.0001) (Fig. 7d), suggesting that specific IgE antibodies might play a crucial action in TsCB-induced rapid worm expulsion from the gut.

Immune protection of rTsCB immunization against larval challenge
Compared with PBS control mice, the mice immunized with rTsCB exhibited a 52.81% reduction of intestinal adults at 6 dpi (Fig. 8a) and a 50.90% reduction of muscle larvae at 35 dpi (Fig. 8b) after oral challenge with 300 T. spiralis infective larvae. The in vitro NBL production for 72 h of adult females from rTsCB-immunized mice was significantly inferior to that of control mice (Fig. 8c) (F (2, 27) = 11.153, P < 0.0001). This result showed that the immunization with rTsCB elicited an immune protection against the T. spiralis challenge infection.
The length of adult females collected from rTsCB-immunized mice at 6 dpi was evidently smaller than that from ISA 201 adjuvant or PBS control mice (Figs. 9, 10) (F (2,27) = 19.390, P < 0.0001); but the length of adult males did not show statistically significant difference among the three groups (F (2, 27) = 1.849, P = 0.177). Moreover, the length of NBL produced by the adult females in immunized mice was clearly shorter than that from the PBS group (F (2,27) = 24.788, P < 0.0001). The muscle larva length from immunized mice was also significantly shorter than that of control mice (F (2, 87) = 68.216, P < 0.0001). These results indicate that the immune response elicited by immunization with rTsCB also hampered the parasite growth and development, reduces the female reproductive capacity, as a result, alleviate the muscle larva burdens in immunized mice.

Discussion
In the present study, the complete cDNA sequence of the TsCB gene was cloned, expressed, and its biological characteristics were investigated. The full-length TsCB sequence was 1071 bp encoding a 40.23 kDa protein.
The amino acid sequences of the TsCB gene had 98% identity with the cathepsin B of six encapsulated species/gene types of the genus Trichinella (T. nativa, T. murrelli, T6, T8, T. britovi and T. nelsoni). Our results demonstrated that rTsCB were expressed in E. coli, with a molecular weight of approximately 40.23 kDa identical to the expected size. After being purified, rTsCB had strong immunogenic properties. Western blot results showed that native TsCB in somatic proteins of muscle larvae, IIL1, 3-day-old adults and NBL were identified by anti-rTsCB antibodies, but not in muscle larval ES protein, indicating that the TsCB is one somatic protein of this nematode, but not a secretory protein of muscle larvae. In the present study, TsCB transcription and expression were also investigated using RT-PCR and IIFA. RT-PCR results indicated that the TsCB gene was transcribed in the four T. spiralis life-cycle stages (muscle larva, IIL, adult and NBL). TsCB expression was detected by IIFA for all T. spiralis life-cycle stages, immunofluorescence staining was located in the cuticle, stichosome and intrauterine embryos of this parasitic nematode, suggesting that TsCB as a surface protein might play a role during larval intrusion of the host's small intestinal epithelium [30,58]. Surface proteins of T. spiralis intestinal stage worms are exposed directly to the host's enteral milieu and local mucosal immune system, they are the important antigenic molecules, and can play a key role  in larval intrusion and development [23,24]. Previous studies have shown that recombinant T. spiralis surface proteins (nudix hydrolase, serine protease, cysteine protease, etc.) participate in larva penetration of intestinal epithelia [15,19,41]. Our previous study demonstrated that when the in vitro larva invasion experiment was performed, rTsCB promoted larva invasion of enterocytes, whereas rTsCB-specific antibodies suppressed larva invasion, this promotion or suppression was dose-dependent of rTsCB or rTsCB-specific antibodies. Silencing TsCB using RNAi significantly impeded the larva invasion (Han et al., unpublished data). The present study suggests that TsCB plays a major part on intestinal mucosal intrusion by this intracellular parasitic nematode.  Vaccination of mice with rTsCB elicited a specific Th2predominant (higher level of IgG1) antibody response to rTsCB. The intestinal and muscle worm reduction observed in the present study is parallel with that of mice vaccinated with recombinant T. spiralis serine proteases [8], nudix hydrolase [45,59] and glutathione S-transferase [42]. The immune protection induced by vaccination with rTsCB may be related to the generation of high levels of serum anti-TsCB IgG antibodies, which neutralized the capacity of cathepsin B to degrade enteral epithelium and other tissues of hosts [20]. Anti-Trichinella IgG may also bind to the epicuticle of enteral IIL1 larvae and generate an antigen-antibody complex in the larva anterior end, which may physically prevent parasite contact from intestinal epithelium cells, thus protect the intestinal epithelium from larval invasion [56,60]. Antibodies against a cathepsin B-like protease (Ac-cathB-1) of Angiostrongylus cantonensis inhibited the L3 larva invasion of the intestines in rats [61]. In addition, previous studies indicated that anti-Trichinella IgG destroyed T. spiralis NBL and ML through an ADCC pattern [53,62,63].
In the present study, the level of anti-TsCB IgE serum in vaccinated mice was also determined. The results showed that vaccination with rTsCB elicited specific IgE, which plays a major role in the rapid expulsion of intestinal infective larvae and adult worms from the guts of vaccinated animals and in delaying larva invasion of intestinal epithelium after oral infection [64,65]. Specific IgE is transported from the blood and exerts an active role in enteral lumen. The IgE combines with the worm surface of T. spiralis and mediates mast cell degranulation to prevent invasion [66,67]. Moreover, IgE also plays an important action in destroying NBL by an antibodydependent cellular cytotoxicity (ADCC) mode [68]. Our results demonstrated that vaccination with rTsCB  a high level of TsCB-specific IgG and IgE antibodies, which resulted in a significant reduction of worm burdens in the intestine and skeletal muscles of rTsCBvaccinated mice. The results suggest that specific IgG and IgE antibodies are crucial for protective immunity against a T. spiralis challenge infection [69].
Additionally, our results also revealed that the length of female adults recovered from immunized mice and the female reproductive capacity (NBL production/female in vitro for 72 hours) was obviously lower than that of the ISA 201 adjuvant or PBS control mice. Length of NBL produced by females from immunized mice was significantly shorter than that of the ISA 201 and PBS groups. These results suggest that immune responses elicited by immunization with rTsCB also impeded intestinal worm growth, and declined the fecundity [56,70]. A decline in female reproductive capacity might be related with females becoming shorter, since the uterus length has a correlation with female fecundity [71].
Trichinella spiralis is a multicellular parasite and its life-cycle is complicated. Different T. spiralis life-cycle stages have stage-specific antigens [72]. Vaccination with an individual Trichinella protein molecule only induced partial protective immunity against challenge. Therefore, oral polyvalent vaccines against diverse T. spiralis stages need to be developed [16,44].

Conclusions
TsCB was expressed in diverse life-cycle stages of T. spiralis and primarily located in the cuticle and stichosome of this intracellular parasite. Vaccination of mice with rTsCB elicited highly specific IgG and IgE responses and partial immune protection, as demonstrated by a significant worm burden reduction in the intestines and muscles of vaccinated mice after oral challenge with T. spiralis infective larvae. The humoral immune responses generated by immunization with rTsCB also impeded intestinal worm growth and declined its fecundity. The results show that TsCB might be considered as a novel potential molecular target to develop vaccines against T. spiralis infection.