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Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

ABSTRACT: Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 ?g) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-? and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 ?g) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

SUBMITTER: Zhao P

PROVIDER: S-EPMC7943400 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Zhao Pengfei P Wang Chaofei C Ding Jun J Zhao Chengfeng C Xia Yingjun Y Hu Yanli Y Zhang Li L Zhou Yanqin Y Zhao Junlong J Fang Rui R

Parasitology research 20210310 5

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewis ...[more]

PMID: 33689009

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Project description:Enterococcus faecalis MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the Eimeria tenella microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant E. faecalis were recorded. The immunized chickens were challenged with 4.0 × 104 E. tenella-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of E. faecalis, respectively. Oral immunizations with recombinant E. faecalis, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (P < 0.01). The groups immunized with four recombinant E. faecalis showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic E. faecalis delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of Eimeria infection. IMPORTANCE Avian coccidiosis caused by Eimeria brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time, Eimeria infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot. Eimeria parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens. Eimeria tenella microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the Eimeria invasion. Enterococcus faecalis is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by E. faecalis, respectively. Oral immunizations were performed to investigate immune protective effects against Eimeria infection.

Project description:Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles carrying recombinant TA4 protein of E. tenella (rEtTA4) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 (PLGA-rEtTA4) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8+ T lymphocytes. However, no significant differences were observed in the proportion of CD4+ T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella. Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles-immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8+ T lymphocytes compared with rEtTA4 subunit vaccine-immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.

Project description:BackgroundChicken coccidiosis, a disease caused by seven species of Eimeria (Apicomplexa: Coccidia), inflicts severe economic losses on the poultry industry. Eimeria tenella is the one of the most virulent species pathogenic to chickens. Many parasitic protozoans are parasitised by double-stranded (ds) RNA viruses, and the influence of protozoan viruses on parasitic protozoans has been extensively reported. E. tenella RNA virus 1 (Etv) was identified in E. tenella, and the complete genome sequence of Etv was analysed. Here, we screened Etv-RNA-dependent RNA polymerase (RDRP)-interacting host protein E. tenella ovarian tumour (OTU) protein-like cysteine protease (Et-OTU) using a yeast two-hybrid system with pGBKT7-RDRP plasmid serving as bait. A previous study demonstrated that Et-OTU could regulate the telomerase activity of E. tenella, indicating that Et-OTU affects E. tenella proliferation. However, whether Etv-RDRP affects the molecular biological characteristics of E. tenella by interacting with OTU remains unclear.ResultsWe obtained seven positive clones from the initial screen, and six of the seven preys were identified as false-positives. Finally, we identified an RDRP-associated protein predicted to be an E. tenella OTU protein. A α-galactosidase assay showed that the bait vector did not activate the GAL4 reporter gene, indicating no autoactivation activity from the RDRP bait fusion. Pull-down and co-immunoprecipitation assays verified the interaction between Et-OTU and Etv-RDRP both intracellularly and extracellularly. Additionally, Et-OTU was able to deconjugate K48- and K6-linked di-ubiquitin (di-Ub) chains in vitro but not K63-, K11-, K29-, or K33-linked di-Ub chains. The C239A and H351A mutations eliminated the deubiquitinase (DUB) activity of Et-OTU, whereas the D236A mutation did not. Additionally, when combined with RDRP, the DUB activity of Et-OTU towards K48- and K6-linked chains was significantly enhanced.ConclusionEtv-RDRP interacts with Et-OTU both intracellularly and extracellularly. Etv-RDRP enhances the hydrolysis of Et-OTU to K6- or K48-linked ubiquitin chains. This study lays the foundation for further research on the relationship between E. tenella and Etv.

Dataset Information

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Publications

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

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