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Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice.

ABSTRACT: Alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by the larval stage of Echinococcus multilocularis. The identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Echinococcus infection. Here, we identified that E. multilocularis calreticulin (EmCRT), a ubiquitous protein with a Ca²⁺-binding ability, could be recognized by the sera of mice infected with E. multilocularis. The native EmCRT was expressed on the surface of E. multilocularis larvae as well as in the secreted products of metacestode vesicles and protoscoleces (PSCs). The coding DNA for EmCRT was cloned from the mRNA of the E. multilocularis metacestode vesicles and a recombinant EmCRT protein (rEmCRT) was expressed in E. coli. Mice immunized with soluble rEmCRT formulated with Freund's adjuvant (FA) produced a 43.16% larval vesicle weight reduction against the challenge of E. multilocularis PSCs compared to those that received the PBS control associated with a high titer of IgG, IgG1 and IgG2a antibody responses as well as high levels of Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-4, IL-5 and IL-10), produced by splenocytes. Our results suggest that EmCRT is an immunodominant protein secreted by E. multilocularis larvae and a vaccine candidate that induces partial protective immunity in vaccinated mice against Echinococcus infection.

SUBMITTER: Chen L

PROVIDER: S-EPMC9610995 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice.

Chen Lujuan L Cheng Zhe Z Xian Siqi S Zhan Bin B Xu Zhijian Z Yan Yan Y Chen Jianfang J Wang Yanhai Y Zhao Limei L

Tropical medicine and infectious disease 20221001 10

Alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by the larval stage of Echinococcus multilocularis. The identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Echinococcus infection. Here, we identified that E. multilocularis calreticulin (EmCRT), a ubiquitous protein with a Ca2+-binding ability, could be recognized by the sera of mice infected with E. multilocularis ...[more]

PMID: 36288020

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Project description:The cestode E. multilocularis causes the disease alveolar echinococcosis (AE) in humans. The continuously proliferating metacestode (larval stage) of the parasite infects mostly the liver and exhibits tumor-like growth. Current chemotherapeutical treatment options rely on benzimidazoles, which are rarely curative and have to be applied daily and life-long. This can result in considerable hepatotoxicity and thus treatment discontinuation. Therefore, novel drugs against AE are urgently needed. The anti-malarial mefloquine was previously shown to be active against E. multilocularis metacestodes in vitro, and in mice infected by intraperitoneal inoculation of metacestodes when administered at 100 mg/kg by oral gavage twice a week for 12 weeks. In the present study, the same dosage regime was applied in mice infected via oral uptake of eggs representing the natural route of infection. After 12 weeks of treatment, the presence of parasite lesions was assessed in a liver squeeze chamber and by PCR, and a significantly reduced parasite load was found in mefloquine-treated animals. Assessment of mefloquine plasma concentrations by HPLC and modeling using a two-compartment pharmacokinetic model with first-order absorption showed that >90% of the expected steady-state levels (Cmin 1.15 mg/L, Cmax 2.63 mg/L) were reached. These levels are close to concentrations achieved in humans during long-term weekly dosage of 250 mg (dose applied for malaria prophylaxis). In vitro structure-activity relationship analysis of mefloquine and ten derivatives revealed that none of the derivatives exhibited stronger activities than mefloquine. Activity was only observed, when the 2-piperidylmethanol group of mefloquine was replaced by an amino group-containing residue and when the trifluoromethyl residue on position 8 of the quinoline structure was present. This is in line with the anti-malarial activity of mefloquine and it implies that the mode of action in E. multilocularis might be similar to the one against malaria.

Project description:BACKGROUND: Regular reformulation of currently available vaccines is necessary due to the unpredictable variability of influenza viruses. Therefore, vaccine based on a highly conserved antigen with capability of induction of effective immune responses could be a potential solution. Influenza matrix protein-2 (M2) is highly conserved across influenza subtypes and a promising candidate for a broadly protective influenza vaccine. For the enhancement of broad protection, four tandem copies of consensus M2 gene containing extracellular (ED) and cytoplasmic (CD) without the trans-membrane domain (TM) reconstituted from H1N1, H5N1 and H9N2 influenza viruses were linked and named as 4sM2. The construct was effectively expressed in Escherichia coli, purified and proteins were used to immunize BALB/c mice. Humoral and cell-mediated immune responses were investigated following administration. RESULTS: Mice were intramuscularly immunized with 4sM2 protein 2 times at 2 weeks interval. Two weeks after the last immunization, first humoral and cell mediated immune response specific to sM2 protein were evaluated and the mice were challenged with a lethal dose (10MLD??) of divergent subtypes A/EM/Korea/W149/06(H5N1), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses. The efficacy of 4sM2 was evaluated by determining survival rates, body weights and residual lung viral titers. Our studies demonstrate that the survival of mice immunized with 4sM2 was significantly higher (80-100% survival) than that of unimmunized mice (0% survival). We also examined the long lasting protection against heterosubtype H5N2 virus and found that mice vaccinated with 4sM2 displayed 80% of protection even after 6 months of final vaccination. CONCLUSION: Taken together, these results suggest that prokaryotic expressed multimeric sM2 protein achieved cross protection against lethal infection of divergent influenza subtypes which are lasting for the long time.

Dataset Information

Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice.

Publications

Immunization with <i>Em</i>CRT-Induced Protective Immunity against <i>Echinococcus multilocularis</i> Infection in BALB/c Mice.

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