Prediction And Identification Of H-2kk-restricted Epitopes In Mouse Hepatitis Virus Strain 3

Methods:1. The H-2KK-restricted CTL epitopes were predicted with the method of supermotif combined with quantitative motif and artificial neural network. The molecular simulation was used to stimulate the space conformation of peptides binding with MHC molecule. The parameters were analysised to further enhance the accuracy of prediction.2. The selected the four top score mouse hepatitis virus strain 3 peptides to be synthesized, purified and identified in terms of molecular weight.3. The analysis on the binding of the synthesized candidate peptides with H-2KK molecules was carried out based on the feature of T2-KK cells.4. we subcutaneously immunized mice by those peptide emulsified in IFA vaccine for three times. The spleen lymphocyte was drawn as effector cells. The LDH assay was carried out to study the killing activity of the mouse hepatitis virus-specific CTLs on target cells expressing H-2KK. In addition, IFN-γreleased of effector cells was measured by ELISPOT method.Results:1. The 4 highest score mouse hepatitis virus epitopes predicted by supermotif combined with quantitative motif and artificial neural network were Mhv141-148(IEPYNGVI),Mhv306-313(YELSGYTV),Mhv228-235(FSVYIGDI),Mhv101-108(NDGIFAKV). The amino acid sequence of the MHV-3 spike proteins was predicted for octapeptides with anchor residues according to the peptide-binding motif of H-2KK, all of the four potential CTL epitopes were suitable to the criteria of H-2KK-restricted CTL epitopes.2. The candidate epitope peptides validated by epitope prediction and molecules modeling methods were then synthesized on a solid-phase simultaneous multiple peptide synthesizer based on the Fmoc strategy, and purified by C18 reverse-phase HPLC to a purity of >95%.3. The analysis on the binding of the synthesized candidate peptides with H-2KK molecules was carried out based on the feature of T2-KK cells. The results showed that among the four candidate epitope peptides, Mhv141-148(IEPYNGVI) and Mhv306-313(YELSGYTV) might have more probability to elicit effective epitope-specific CTLs. 4. C3H/Hej mice were immunized thrice (with a 7-day interval) by s.c. injection with various peptides emulsified in Freund’s Incomplete adjuvant, Mice were sacrificed, and spleens were removed 21 days after immunization, splenocytes were used as effector cells. A standard LDH assay was arried out to study the killing activity of the mouse hepatitis virus-specific CTLs on target cells expressing H-2KK. These results indicated that the immune response induced by mouse hepatitis virus epitopes was specific and restricted by H-2KK. ELISPOT test showed that the mouse hepatitis virus-specific epitopes were capable of enhancing IFN-γrelease effectively.Conclusions:1. There are two H-2KK-restricted CTL epitopes screened out from the full-length amino acid sequence of mouse hepatitis virus using method of supermotif combined with quantitative motif and artificial neural network. They are Mhv141-148(IEPYNGVI) and Mhv306-313(YELSGYTV), which were determined for the first time.2. The experiments in vitro and in vivo prove that the above mouse hepatitis virus epitopes can effectively elicit mouse hepatitis virus-specific CTLs against mouse hepatitis virus positive and H-2KK matched target cells. These data suggest that mouse hepatitis virus peptide vaccine can induce immunity against target cells in a H-2KK restricted fashion.3. The in vivo experiments prove that mouse hepatitis virus-specific epitopesMhv141-148(IEPYNGVI) and Mhv306-313(YELSGYTV) can enhance IFN-γreleasing effectively, which indicated that mouse hepatitis virus-specific epitopes not only elicit a specific immune response, but also elicit a non-specific immune response.In conclusion, the above studies show for the first time that the mouse hepatitis virus-specific epitopes Mhv141-148(IEPYNGVI) and Mhv306-313 (YELSGYTV) are capable of eliciting a specific anti-virus immune response by mouse hepatitis virus-specific CTLs, as well as a non-specific anti-virus immune response by enhancing cytokines releasing. These finding show that mouse hepatitis virus-specific epitopes vaccines have various advantages such as broad-spectrum, high-effect, high specificity and safety, which will provide theoretical evidence for their clinical application, These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.