The Establishment And Identification Of Humanized MHC Transgenic Mice And Basic Application Research

Animal models play critical roles in pre-clinical researches such as disease pathogenesis, development and evaluation of vaccines and drugs, while there are still several limitationsin current animal models. The states of physiological and immunological in murine and nonhuman primates are district from that of mankind radically, especially the MHC restriction, cannot eliminate the influence of species specificity in pre-clinical experiments to reflect the immune characteristics of humans accuratly. Besides, NOD/SCID mice have some application in transplantation and tumor researches. Owing to the different restriction between HLA and H-2, there exist the defects like immunologic rejection and low efficiency of transplantation. Accordingly, how to construct ahigher lever of ―humanized‖ transgenic mouse strain which could mimic human immune responses more accuracy and reliable has became the emphasis in humanized animal models.Major histocompatibility complex(MHC) molecule is the most polymorphic gene group among human and mammalians. MHC plays a vital role in the positive and negative selection of T lymphocytes in thymus, facilitating the differentiation and maturation of lymphocytes, activating and regulating the adaptive and innate immune system. The structure and function of HLA and H-2 is quite similar, while their MHC restricted function is different. The MHC restriction differs markedly according to the geographical region and ethnicity. The dominant MHC genotypes in China are different from that in European, American, Middle East and other regions. HLA-A2, A11 and A24 account for 80% in China, while HLA-A2, A1 and A3 are the higher frequency genotypes in European countries. In HLA class II molecular, HLADR09, DR15 and DR01 account for 70% in China, and HLA-DR01 as well as HLA-DR03 and HLA-DR04 are the main alleles in Europe.HLA molecular replaces H-2 in MHC class-I or II transgenic mice, and eliminate the competitive inhibition of endogenous H-2 in immune responses, resulting in a more accurate and efficient reflect on HLA in humans. In this study, we generated a new homozygous HLAA11/DR1 Tg mouse stain(HLA-A11+/+/DR1+/+/H-2-β2m-/-/IAβ-/-) which carried the Chinese dominant HLA class I and II alleles, which applied in the the evaluation of vaccine, and to screen and identify the HLA restricted CTL epitopes of pathogens, and to design and optimize the novel epitopes-based vaccines. Meanwhile, we constructed a immuno-deficient MHC transgenic mice(HLA-A2+/+/DR1+/+/H-2-β2m-/-/IAβ-/-/Rag2-/-/IL-2rγ-/-/Perf-/-)and a hPBMCtransplantation mouse model, and provide technical support of pathogen infection models, tumors, transplantation mechanism, and the invention of vaccines and drugs. 1. The generation and identification of homozygous HLA-A11/DR1(HLAA11+/+/DR1+/+/H-2-β2m-/-/IAβ-/-) Tg mice and the application of this novel mouse model into the development and evaluation of vaccines 1.1 The generation and identification of homozygous HLA-A11/DR1 Tg mice. A chimeric HLA-A*1101 monochain fragment encompassed HLA-A11 leader sequence, α1 and α2 domains, murine H-2Db(from the α3 domain to the COOH terminus), and human β2m, which was covalently linked between the leader sequence and the 5‘ end of HLA-A11(α1 and α2) domains via a 15 aa linker encoding Gly4Ser3. The purified HLA-A11 fragment was microinjected into pronuclei from fertilized(C57BL/6 × C57BL/6) F1 mouse eggs to generate transgenic embryos. The embryos were then re-implanted into pseudopregnant female mice. The HLA-A11 founder mice showing the highest expression of HLA-A11 were then crossed with A2/DR1 mice. After a series of backcrossing, HLA-A11/DR1 Tg mice(HLAA11+/+/DR1+/+H-2-β2m-/-/IAβ-/-) were generated. The HLA transgenes and H-2 knockout mice were identified by PCR, the genetic stability of the offsprings was confirmed. The expression of HLA tranegenes in splenocytes of HLA-A11/DR1 mice was stronger than C57BL/6 and H-2-I/II knockout mice. Flow cytometric results showd that the numbers of m CD3+CD4+ and mCD3+CD8+ T cells in HLA-A11/DR1 mice were comparable with WT mice. These results showed that the HLA transgenes enabled the differentiation and maturation of T lymphocytes in A11/DR1 mice. HLA-A11/DR1 mice could mount HLA-A11 restricted responses after immunized two known A11-restricted epitopes derived from HIV-Pol177-188 and HIVGP16032-45. Results showed that HLA-A11/DR1 mice could mount the HLA-A11-restricted, epitope-specific, IFN-γ-producing mCD8+ T cellular immune response. 1.2 The application of HLA-A11/DR1 mice into the evaluation and development of vaccines(1)The application of HLA-A11/DR1 mice into the evaluation of immune responses of vaccines. To examine the immune responses to vaccine candidates and commercial vaccines, HLA-A11/DR1 mice were immunized with HIV-MEP1 and a recombinant HBsAg vaccine. ELISA test revealed an increase(p<0.05) in the levels of HIV-MEP1-specific IgG antibodies in A11/DR1 mice, and they also secreted more IFN-γ than C57BL/6 mice. The amount of HBsAg-specific IgG antibodies of A11/DR1 mice was similar to that of C57BL/6 mice. It is worth noting that HLA-A11/DR1 mice generated 4-5-fold more IFN-γ than WT mice, indicating a satisfactory cellular immune response and ability to mimic human cytotoxic response, which demonstrated that the novel mouse model have the capability to estimate the immune responses induced by commercial vaccines and novel vaccine candidates.(2)Based on the HLA-A11/DR1 mice model to predict and screen the HLA-A11 restrcted CTL epitopes of EBOV and MERS-CoV, further to stuty the epitopes-based vaccines. In order to predict HLA-A11 restricted CTL epitopes of EBOV and MERS-CoV, we analyzed the sequence of amino acid of EBOV-GP and MERS-CoV by bio-information online software SYFPEITHI and NetMHC, next synthesized the 24 HLA-A11 potential peptides which got the higher binding scores for further verification. ELISPOT results showed that the HLA-A11 specific CTL responses were stimulated by two epitopes from EBOV-GP(EBOV-GP76-84 and EBOV-GP423-431)and four from MERS-CoV-S(MERSS152-160、MERS-S720-728、MERS-S889-897 and MERS-S1092-1100). These candidate epitopes have the potential to contribute to the design of multi-epitopes based vaccines of highly-risk pathogens. 2. The construction of immuno-deficient humanized MHC transgenic mice "HUMAMICE" and the characteristics of hPBMC-engrafted-HUMAMICE 2.1 The construction and immuno-deficiency characteristics of HUMAMICE(1)The construction and identification of HUMAMICE. The immuno-deficient MHC transgenic humanized HUMAMICE(HLA-A2+/+/DR1+/+/β2m-/-/IAβ-/-/Rag2-/-/IL2rγ-/-/Perf-/-mice) were generated on the background of HLA-A2/DR1 mice(HLA-A2+/+/DR1+/+/β2m-/-/IAβ-/-) by several inter-crossing with Rag2-/- mice, IL2rγ-/- mice and Perf-/- mice into homozygosity. The obtained final homozygotes HUMAMICE were checked by specific PCR and FACS. There only a residual population of 0.4% CD3+CD4+ T, 0.4% CD3+CD8+ T, and 0.1% CD19+CD20+ B cells were found in HUMAMICE. The absence of murine T and B cells is an important criterion for the immuno-deficient status of highly immuno-deficient mice.(2)The analysis of immuno-deficiency characteristics in HUMAMICE. The structure of spleen and and thymus of HUMAMICE is not complete. To confirm the immunodeficiency status of HUMAMICE, we engrafted subcutaneously human RAMOS tumor cells into HUMAMICE and A2/DR1 mice. Tumors were not able to grow in A2/DR1 mice whereas they induced tumors in HUMAMICE. Furthermore, to verify that the inability of HUMAMICE to reject RAMOS cells is due to lack of the mouse-derived immune system, we transferred the splenocytes, purified CD8+ T lymphocytes or other lymphocytes derived from A2/DR1 mice which had rejected RAMOS cells into HUMAMICE and then developed a solid tumor. Altogether, these results confirm that HUMAMICE are severely deficient. 2.2 The hPBMC engraftment in immuno-deficient humanized HUMAMICE(1)The exnogenic hPBMCs transplantation in HUMAMICE. PBMC were collected from healthy volunteers, HLA-A2+ individuals were identified by flow cytometry and HLADR1+ individuals were examined by specific PCR. HUMAMICE were irradiated the day before transfer and followed given by intravenous injection of hPBMCs. Mice were monitored weight and clinical symptoms twice per week. None of HUMAMICE showed no wight loss in 60 days after the transplantation or developed GvHD signs.( 2) The immune reconstitution characteristics in HUMAMICE after hPBMC engraftment and HBsAg immunization. In order to evaluate the engraftment in this novel model, we transferred HLA-matched(HLA-A2+/DR1+) hPBMCs into HUMAMICE. 10 days after transplantation, HUMAMICE received three i.m. injections of HBsAg. The functional ability of the adoptively transferred hPBMCs inHUMAMICE respond to HBsAg was evaluated, a significant HBsAg-specific-h IgG response was amplified after recall immunization boost. The HBsAg-specific-hIgM responsewas detected after the primary-immunization. Flow cytometry analysis was done five and eight weeks after transferred. Results of two representative mouse(n=9) were shown that 11.6% and 9.70% were composed by hCD45+ cells, with 45.2% and 49.4% of this population were hCD45+h CD19+, and 17.1% and 16.1% of hCD45+ were T lymphocytes(hCD45+hCD3+): 29.0% and 30.0% of them were CD8+T cells(hCD3+hCD8+) and approximately 60.9% and 59.0% were hCD3+hCD4+ T lymphocytes. Above all, after the hPBMC transferred, the immune system of HUMAMICE was reconstructed. 3. Conclusion:(1)The Chinese HLA dominanted HLA-A11/DR1(HLA-A11+/+/DR1+/+/H-2-β2m-/-/IAβ-/-) transgenic mice strain were established. This novel mouse strain possesses the HLA-restricted characteristic and a normal ability to reponse to the antigens. The evaluation of candidate vaccine and commercial vaccine showed that it could apply in to the evaluation of vaccines. Furthermore, we sceened two HLA-A11 restricted epitopes from EBOV GP protein and four from MERS-CoV S protein. Above all, the novel HLA-A11/DR1 Tg mice could facilitate the screen and identification of Chinese dominant HLA-restricted CTL and Th epitopes of important pathogens, and provide a new promising technical tool to the immunological mechanism as well as the invention and evaluation of vaccines.( 2) The novel immuno-deficient HLA transgenic mice "HUMAMICE"(HLAA2+/+/DR1+/+/β2m-/-/IAβ-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice) were established, which expressed human HLA molecules instead of murine H-2, and represent no lymphocytes in their immune system, the development of spleen and thymus is not complete. And whose immuno-deficient status was reversed by transferring the functional HLA-matched(HLA-A2+/DR1+) hPBMCs and then producing the mice with an immuno-competent status with a functional human immune system. We showed that in this particular HLA-matched context, the hPBMCtransfer led to high lymphocytes engraftment rates without GvHD over three months in this novel model. Furthermore, to evaluate the abilityof transferred human cells, we immunized hPBMC-HUMAMICE with HBsAg vaccine which resulted in robust and reproducible high level of humanized HBsAg-specific IgM and IgG antibodies, implying that both transferred T and B lymphocytes were functional. In conclusion, these findings indicated that the hPBMCsHUMAMICE model represents a promising model to dissect human immune responses in various human diseases, including pathogens, infectious diseases, cancers and tumors, which could facilitate the development of novel vaccines, drugs and cellular therapies.