Improving The Immunogenicity Of An EtMIC2 Protein Against E. Tenella By Random Mutagenesis

Avian coccidiosis, caused by obligate intracellular protozoan parasites of the genus Eimeria, is one of the most important diseases which are estimated to be in excess of $3 billion in the world and ￥3 billion in China per annum with costs of prevention and treatment. Current prevention of coccidiosis rely mainly on drugs and vaccine, with the development of drug resistance and more regulations on the use of anticoccidial drugs, vaccination is urgently needed to control the chicken coccidiosis. Almost all of the available anticoccidial vaccines on the market are based on varied combinations of multiple live Eimeria species currently. The safety of wild-type vaccines and inherent production limitations of attenuated vaccines prompt the researchers pay more attention to subunit anticoccidial vaccines.The development of anticoccidial subunit vaccines relies on appropriate antigens with high antigenicity and immunogenicity. The identification of anti-parasite vaccine candidates is difficult due to the complex life-cycle, identification of genomes and screen of the antigen immunogenicity impeded the development of subunit vaccines against parasites. Many efforts to improve protective efficacy of antigens have been made which included:novel adjuvants; improving vectored of vaccine; inducing of non-specific immunization by probiotic. But these approaches could not improve the immunogenicity. Consequently, improving the found vaccine candidate could be a rapid and effective method.Directed evolution is widely used in the field of protein engineering to develop and identify novel protein variants, and one important application of this technology is the generation of improved vaccine immunogens. In general, amino acid substitution may have profound effects on immune response and recognition, such as reversing inhibition of host innate immune response pathways, impairing T-cell activation, and improving protein immunogenicity. Random mutagenesis is one of the most common and successful methods of direct evolution.MICs are essential for motility of the parasite, binding of the host cell-surface proteins, invasion of host cells and intracellular survival. The E. tenella microneme-2 protein expressed in all the life stages of E. tenella is a member of MICs. The protein is abundant within the microneme organelles when parasite invades host cell and is copiously secreted onto the host cell surface. Previous studies have shown that immunization of chickens with the EtMIC2 gene or recombinant proteins could provide partial immunity protection. We brought mutation evolution into the EtMIC2 gene using random mutagenesis and generated a variant gene library. Then the variant protein library was displayed on the yeast surface. After sorting of screening with FACS and immunity evaluating to experimental infection, variants were generated for the development of coccidian vaccines.1. The construction of an EtMIC2 variant gene libraryTo generate a mutagenesis gene library that introduced mutations into the wild-type EtMIC2 gene, epPCR was carried out with the recombined plasmid T1-EtMIC2 as a template. About 33μg epPCR production were used for cloning with the plasmid vector T1 and then transformed into Top 10 cells. Finally approximately 107 independent colonies were grown on 323 ampicillin resistant agar plates.5782 positive clones of all plate were identified using PCR amplification from bacterial liquid and the positive rate showed 89.5%. The results indicate that an EtMIC2 variant gene library was constructed. To analyze the positions and numbers of the mutation in the EtMIC2 variant gene, plasmids were isolated from the cells. Sequences of 21 insert fragments were determined and the results showed that the novel EtMIC2 variant genes contained about average 6.86±3.21 point mutations in random position of 951 bp. After the mutagenesis genes were translated, average 4.19±2.68 aa mutations were been introduced into 21 variant proteins.2. The construction of an EtMIC2 variant protein libraryThe mutagenesis gene library recombined with pCTCON2 vector was transformed into the S. cerevisiae EBY100 strain. After hundreds of transformation, approximately 108 yeast clones were grew on SD-CAA agar plates. The proteins displayed on yeast surface were detected using primary antibody of specific anti-EtMIC2 polyclonal antibody and FITC-conjugated goat anti rabbit secondary antibody, then fluorescence was observed by fluorescence microscopy. The western blot assay to test the EtMIC2 variant protein displayed on the yeast surface was carried out with 100 mM DTT. The results indicate that EtMIC2 variant proteins library were displayed on surface of yeasts.3. Screening from EtMIC2 variant protein library by FACSThe cell sorting experiments were performed on a BD FACSAria Cell-Sorting System with gate settings for the strongest (PI), moderate (P2) and weakest (P3) fluorescent signal based on the affinity between polyclonal antibody and antigen. The mutant library was subjected to thrice consecutive sorting times of FACS selection independently and amplification on SD-CAA agar plates. Finally 401 cells from there gate were sorted respectively from 108 yeast cells. A total of 19 clones of EtMIC2-displaying variant were gotten from SD-CAA agar plates. The EtMIC2 variant genes were amplified using recombinant plasmids extracted from these yeast cells as the DNA templat. After sequencing, the results showed that 5 proteins had no aa point mutation including one protein of P2, and four proteins from P1.6 proteins appeared termination codon after random mutagenesis.2,3, 9,4,7,14 and 11 mutational amino acids were introduced into 2218,2120,2221,1225,1130, 2118 and 2119 proteins respectively.19 yeast clones were also detected by the immunofluorescent labeling assay and flow cytometry method. Finally these seven mutational proteins were selected to detect the immunogenicity and protective efficacy against chicken coccidiosis.4. Evaluation of immunoprotective effect against E. tenella infection.EtMIC2 and seven mutational genes were cloned in pET30a plastids and transformed into BL21 (DE3). After expression and purification, the proteins were detected using Western blot. EtMIC2 variant proteins were emulsified with Freund’s adjuvant used as subunit vaccine. Birds were given twice injection with proteins at 7 and 14 days. At 7 d post-secondary immunization, birds were given 30,000 oocysts except PBS-I group by oral gavage. The result of ACI indicated 1130 and 2119 proteins can provided moderate activity against 30,000 oocysts of E. tenella infection. Almost all immunized birds showed significantly slight (P< 0.05) on body weight gains, fecal oocyst shedding and caecum lesion compared to mock-immunized (PBS) challenged birds groups. Especially, compared to the wild-type EtMIC2 controls, lesion scores of 1130,2119 and 2118 protein groups and oocyst shedding of 1130 protein group displayed significantly different (P< 0.05). The lympHocytes from groups vaccined with 2119 and 1130 proteins demonstrated a significant elevated response (P<0.05) to EtMIC2 and EtMIC2 variant proteins compared with the wild-type EtMIC2. Comparison of the wild-type EtMIC2 group,2119 and 1130 groups revealed the significant increase (P <0.05) proportion of CD4+ and CD8+ PBLs on day 10 post-infection. The secreted of sIgA were increased at 10 day P.I., in particular,1130 and 2119 group had the higher level compared to the wild-type protein group.Taken together, these results indicated that the mutational proteins’protective efficacy against chicken coccidiosis had been improved. Two variants (2119 and 1130 proteins) were generated with several amino acid mutations altered the immunological information in peptide epitopes which improve the immunogenicity and protective efficacy. This is the first time that the protein from parasite was used to change immunogenicity by random mutagenesis for vaccination.