Molecular cloning and characterization of a novel peptidase from Trichinella spiralis and protective immunity elicited by the peptidase in BALB/c mice.

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.

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.

Intranasal immunization with recombinant Trichinella spiralis serine protease elicits protective immunity in BALB/c mice.

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.

Protective immunity against Trichinella spiralis in mice elicited by oral vaccination with attenuated Salmonella-delivered TsSP1.2 DNA.

Trichinellosis is a worldwide important food-borne zoonosis caused mainly by ingesting raw or undercooked pork infected with Trichinella spiralis larvae. The development of vaccine is needed for preventing swine from Trichinella infection to ensure pork safety. Previous studies showed that T. spiralis serine protease 1.2 (TsSP1.2) is a vaccine candidate against Trichinella infection. In this study, the complete TsSP1.2 cDNA sequences were cloned into pcDNA3.1, and the rTsSP1.2 DNA was transformed into attenuated Salmonella typhimurium strain ΔcyaSL1344. Oral vaccination of mice with Salmonella-delivered rTsSP1.2 DNA vaccine induced an obvious intestinal mucosal IgA response and a systemic Th1/Th2 immune response; the vaccinated mice showed a 33.45% reduction of intestinal adult worms and 71.84% reduction of muscle larvae after T. spiralis larval challenge. The protection might be due to the rTsSP1.2-induced production of specific anti-TsSP1.2 sIgA, IgG, IgG1/IgG2a, and secretion of IFN-γ, IL-4 and IL-10, which protected intestinal mucosa from the parasite invasion, inhibited worm development and reduced female fecundity. The results indicate that the attenuated Salmonella-delivered rTsSP1.2 DNA vaccine offers a prospective strategy for the prevention and control of animal Trichinella infection.