The Establishment Of Humanized MHC Transgenic Mice And Reseach On The MHC-Restricted Epitopes In The EBOV-NP Protein

Based on the similar structure and function of the immune system between human and mice,mice models are used to the most human immune system functional study.However,due to the species-specificity against diseases or pathogen infection,etc.,the immune responses process produced by mice and human are significantly different.One reason is the different MHC-restricted molecules,which are known as H-2 in mice and HLA in human.Therefore,the characteristics of the human immune system cannot be effectively reflected in mice models in many studies.And the results of the clinical research cannot reproduce those of the preclinical experimental animals.The construction of humanized mice capable of simulating human immune response whic help to understand the mechanisms of the diseases,the development of vaccines and drugs and make the accuracy of preclinical experimental research results.The MHC molecules are highly polymorphic,and there are different distributions of dominant haplotypes in various populations.People in a group with the same or similar MHC molecules in order to resist environmental changes and pathogen invasion.This maker certain MHC genes are identical in the same race or individuals in the same area,and significant differences from others.Among the MHC class I molecules,HLA-A2,HLA-A11 and HLA-A24 are the predominant subtypes in Chinese.However,HLA-A2,HLA-A3,and HLA-A1 are superiority in Europeans.Among the MHC class II haplotypes,the proportion of HLA-DR15 subtype in Chinese is more than 25%,and the HLA-DR1 subtype is 10%~15%.The predominant subtypes in the population from the European and the Middle East are HLA-DR1,HLA-DR3 and HLA-DR4.The strategy for the establishment of human MHC transgenic mice is to integrate the exogenous HLA genes into the mice genome and knock out endogenous H-2 gene in mice simultaneously.The MHC transgenic mice models can be used effectively in the study of vaccines for human due to the immune responses similar to human arouse after stimulation.In the year of 2014,the largest epidemic of Ebolavirus(EBOV)occurred in Africa.The Ebolavirus belongs to the Filoviridae family,which causes highly mortality in both humans and non-human primates.Despite extensive researches on the EBOV,there are still no effective treatments or vaccines.The traditional vaccinations with whole inactivated or live attenuated microbes may cause inflammation and other serious immune problems.The Ebolavirus nucleoprotein(NP)is a major structural protein,which plays a key role in the virus replication and packaging process,and confers protective immunity.Thus,a desirable vaccine would be one that bears the epitopes from EBOV-NP that can be presented by common MHC class I molecules which combined with biological information technology for prediction and identification of epitopes.The purpose on this study was to construct HLA-A2/DR15 double transgenic mice model with dominant MHC class I and MHC class II genotypes in Chinese.And the epitopes of EBOV-NP for HLA-A11-restriction were predicted and screened with the humanized HLA-A11/DR1 double transgenic mice which were constructed earlier by our group.1.Establishment and Functional Analysis of the MHC(HLA-A2/DR15)Double Transgenic Mice ModelsThe optimized nucleic acid sequence of HLA-DRB1*15:01 gene was integrated into the genome of the mice embryoes by microinjection.The embryoes were then transplanted into a surrogate mother mice and constructed the HLA-DRB15 mice.The founder mice were mating with HLA-A2+/+DR1+/+?2m-/-IA?-/-transgenic mice.The HLA-A2+/+DR15+/+?2m-/-IA?-/-double transgenic mice were generated by self-crossing and backcrossing.The expression of HLA-A2 and HLA-DR15 genes and knockout of mouse H-2 gene in offsprings were identified.The HLA class I and HLA class II genes on the splenocytes' surface were detected by flow cytometry.The results showed that the expression of transgenes in transgenic mice significantly stronger than those in C57BL/6 mice.And the number of m CD8+T cells and m CD4+T cells detected in transgenic mice were relatively similar to that in the C57BL/6 mice,indicating that exogenous HLA molecule can develop mature T cells in transgenic mice and perform the corresponding functions.Detectable IgG antibody responses to EBOV-NP in the Ad5-EBOV-NP immunized transgenic mice by ELISAs showed no significant differences from that the Ad5-EBOV-NP immunized wild-type mice.Results indicated that the transgenic mice can arouse the normal humoral immune responses.Nine epitopes mount HLA-DR15-restricted were selected for screening in our HLA-A2/DR15 transgenic mice.The HLA-A2/DR15 transgenic mice could be also verified to induce T-cells responses.And 6 epitotes induced stronger IFN-? in the splenocytes from Ad5-EBOV-NP immunized HLA-A2/DR15 mice than those from wild mice.Above all,a humanized HLA-A2/DR15 double transgenic mice were established succesfully in the study,which could be applied for epitopes screening,the development of vaccines and drugs,diseases pathogenesis.2.Identification of HLA-A11-restricted CD8+ T-cell epitopes in EBOV-NPThe EBOV-NP gene was first cloned in the p ET-32 a vector and then subcloned into the shuttle vector p Shuttle-CMV.The shuttle vector was then linearized with Pme I and recombined to the BJ5183-AD-1 bacteria,which carry the Ad Easy-1 plasmid that encodes the adenovirus-5(Ad5)genome.Recombinant Ad5 plasmids were designated as Ad5-EBOV-NP.Then the recombinant Ad5 plasmid were digested with Pac I and then used to transfect into the AD-293 packaging cell line.The transfected cells were identified on the basis of the adenovirus-related cytopathic effects.The recombinant Ad5-EBOV-NP viruses were then reamplified in AD-293 cells and purified.To check the expression of the recombinant virus,AD-293 cells were infected with the purified Ad5-EBOV-NP and observed under a fluorescence microscope.Western Blot was used to detect the expression of EBOV-NP protein in Ad5-EBOV-NP infected AD-293 cells.The HLA-A11/DR1 and C57BL/6 mice were immunized three times with Ad5-EBOV-NP or Ad5,and the Ig G antibody responses to EBOV-NP around the third vaccination detected by ELISAs.The results showed the antibody levels were significantly greater in Ad5-EBOV-NP immunized HLA-A11/DR1 transgenic mice than those in Ad5-immunized transgenic mice.Compared to the responses of the splenocytes from the Ad5-immunized transgenic mice,six of the ten peptides,namely,GR-9,VR-9,QR-9,EK-9,PK-9,and RK-9,induced significant IFN-? production by the splenocytes from the Ad5-EBOV-NP-immunized mice.The remaining four peptides did not induce a response.To further verify that epitopes are HLA-A11-restricted,the HLA-A11/DR1 and C57BL/6 mice were immunized three times with Ad5-EBOV-NP.The peptides GR-9,VR-9,EK-9,PK-9,and RK-9 induced strongly CD8+T-cell responses by the Ad5-EBOV-NP immunized transgenic mice compared with the Ad5-EBOV-NP immunized wildtype mice.The QR-9 was not specific HLA-A11 restricted because it could induce IFN-? production by the splenocytes from the Ad5-EBOV-NP immunized mice than those from the immunization of Ad5,no significant difference of IFN-?induced than those from wild-type C57BL/6 mice Thus,these experiments with HLA-A11/DR1 mice identified five novel HLA-A11-restricted CTL epitopes in the text of EBOV-NP protein.In summary,we successfully established HLA-A2/DR15 double transgenic mice with the Chinese dominated HLA molecules.And the new MHC transgenic mice is HLA-DR15 restricted and had the ability to responses to antigens.The HLA-A2/DR15 double transgenic mice will be a candidate model for evaluation of vaccines and drugs used for human.Five HLA-A11-restricted CTL epitopes in the EBOV-NP protein were screened with the HLA-A11/DR1 mice,which could be used for new peptide-based vaccines for EBOV.