Ruo Dan Liu

Phage-displayed specific polypeptide antigens induce significant protective immunity against Trichinella spiralis infection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emerging/re-emerging disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects humans, domestic animals and wildlife. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Phage particles are especially ideal vaccine delivery vehicles because they do not interfere with the immune response against the displayed peptide antigens, and, if anything, are more likely to efficiently direct antigen expression to professional antigen-presenting cells. In this study, Tsp10 polypeptide, which was encoded by a cDNA fragment of Trichinella spiralis intestinal infective larvae and was found to bind to normal mouse intestinal cells, was displayed on the surface of T7 phage. Anti-Tsp10 antibodies were able to recognize the native Tsp10 protein mainly localized to the stichosome of T. spiralis. Mice immunized with the recombinant phage T7-Tsp10 showed a 62.8% reduction in adult worms and a 78.6% reduction in muscle larvae following challenge with T. spiralis muscle larvae. Our results demonstrate that the vaccination with Tsp10 polypeptide displayed by T7 phage elicits the Th2-predominant immune responses and produces a significant protection against T. spiralis infection in mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against T. spiralis.

Identification of Differentially Expressed Genes of Trichinella spiralis Larvae after Exposure to Host Intestine Milieu

Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the ‘tester’. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.

Normal Mouse Intestinal Epithelial Cells as a Model for the in vitro Invasion of Trichinella spiralis Infective Larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.

Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae.

The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens.

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31 kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25-55 kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P>0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P<0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.

Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice

BackgroundWe have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity. The aim of this study was to investigate the immune protection induced by TsNd DNA vaccine.MethodsThe full-length cDNA sequence of TsNd gene was cloned into pcDNA3.1 and used to immunize BALB/c mice by intramuscular injection. Transcription and expression of TsNd were detected by RT-PCR and IFT. The levels of specific IgA, IgG, IgG1 and IgG2a, and cytokines were assayed by ELISA at weeks 0, 6 and 8 post-immunization. The immune protection of TsNd DNA vaccine against challenge infection was investigated.ResultsImmunization of mice with TsNd DNA elicited a systemic Th1/Th2 immune response and a local mucosal IgA response. The in vitro transcription and expression of TsNd gene was observed at all developmental stages of T. spiralis (ML, IIL, AW and NBL). Anti-rTsNd IgG levels were increased after immunization and levels of IgG1 were obviously higher than that of IgG2a. Intestinal specific IgA levels of immunized mice were significantly higher than those of vector and PBS control mice. Cytokine profiling also showed a significant increase in Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice on stimulation with rTsNd. Vaccination of mice with pcDNA3.1-TsNd displayed a 40.44% reduction in adult worms and a 53.9% reduction in larval burden.ConclusionsTsNd DNA induced a mixed Th1/Th2 immune response and partial protection against T. spiralis infection in mice.

Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity

We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice.

Trichinella spiralis: Low vaccine potential of glutathione S-transferase against infections in mice

We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis, but not in ML excretory-secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection.

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

Background Sparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis. Methodology/Principal Findings The full length SeCP gene was cloned, and recombinant SeCP (rSeCP) was expressed and purified. Western blotting showed that rSeCP was recognized by the serum of sparganum-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude or ES antigens. Expression of SeCP was observed at plerocercoid stages but not at the adult and egg stages. Immunolocalization identified SeCP in plerocercoid tegument and parenchymal tissue. The rSeCP had CP activity, and the optimum pH and temperature were 5.5 and 37°C, respectively. Enzymatic activity was significantly inhibited by E-64. rSeCP functions to degrade different proteins and the function was inhibited by anti-rSeCP serum and E-64. Immunization of mice with rSeCP induced Th2-predominant immune responses and anti-rSeCP antibodies had the potential capabilities to kill plerocercoids in an ADCC assay. The sensitivity of rSeCP-ELISA and ES antigen ELISA was 100% when performed on sera of patients with sparganosis. The specificity of rSeCP-ELISA and ES antigen ELISA was 98.22% (166/169) and 87.57% (148/169), respectively (P<0.05). Conclusions The rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.

Proteomic analysis of Trichinella spiralis adult worm excretory-secretory proteins recognized by early infection sera

At the intestinal stage of a Trichinella spiralis (T. spiralis) infection, the excretory-secretory (ES) antigens produced by adult worms (AWs) result in an early exposure to the hosts immune system and elicit the production of specific antibodies; the AW ES proteins might provide early diagnostic markers of trichinellosis. The aim of this study was to identify early serodiagnostic markers from T. spiralis AW ES antigens. T. spiralis AWs were collected at 72h post infection, and their ES antigens were analysed by SDS-PAGE and Western blot. Then, the immunoreactive bands were subjected to shotgun LC-MS/MS and bioinformatics analyses. Our results showed that only one protein band (33kDa) was recognized by the sera of mice infected with T. spiralis at 8 days after infection. The shotgun LC-MS/MS analysis identified 23 proteins that were then clustered into 10 types; these proteins had molecular weights of 28.13-71.62kDa and pI 5.05-9.20. Certain enzymes (e.g., serine protease, adult-specific deoxyribonuclease [DNase] II, peptidase S1A subfamily, and multi cystatin-like domain protein) were found to be highly represented. The functions of the 10 proteins were further analysed: of the 6 annotated proteins, 3 had serine hydrolase activity and 2 had DNase II activity. These results provide a valuable basis for identifying early diagnostic antigens and vaccine candidates for trichinellosis.