인간화마우스 모델을 활용한 Francisella tularensis 항원의 면역반응에 관한 연구

학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2018. 8. 박재학.Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon because of its low infectious dose, and the ability of aerosol transmission. Although an attenuated FT live vaccine strain (LVS) has been developed, this vaccine exhibits side effects and residual virulence. Therefore, subunit vaccines using parts of pathogens have been proposed as an alternative to live vaccines. Tul4 and FopA are outer membrane proteins of FT, and have been reported to play an important role in the bacteriums immunogenicity, which support the potential of subunit vaccines as candidates against FT. Although in vitro and mouse models are standard paradigms for vaccine development, these models have limitations including differences in susceptibility to FT between mice and humans. Humanized mice (hu-mice) bearing the human immune system have been developed to study human-specific diseases, and have been reported to produce human immunoglobulins against pathogens. Therefore, hu-mice have the potential to overcome the limitations of animal models used in clinical research. In this study, we reported that a subunit vaccine constructed based upon outer membrane epitopes, Tul4 and FopA, elicited the human-specific immunity in hu-mice model. The study described in Chapter I reported the in vitro and in vivo immune response of a subunit vaccine comprising the recombinant peptides Tul4 and FopA generated from epitopes on FT outer membrane proteins. Dendritic cells (DCs) stimulated by recombinant peptides with adjuvant CpG oligodeoxynucleotide induced robust immunophenotypic changes in DC maturation and secretion of inflammatory cytokines (interleukin [IL]-6 and IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in mixed lymphocyte reactions. Finally, I investigated in vivo immune responses by assessing antibody production in C57BL/6 mice. Total immunoglobulin (Ig) G was produced after immunization and levels peaked 6 weeks later. Moreover, Tul4-specific IgG was confirmed in mice receiving peptides with or without CpG. Based on these results, I revealed that the recombinant peptides Tul4 and FopA have immunogenicity, and could be a safe subunit vaccine candidate against FT. Chapter II described the immune response of mice—humanized with human CD34+ cells (hu-mice)—to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an LVS, as well as human T cells in the spleen. These results suggest that a cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates. In conclusion, the present study demonstrates the efficacy of recombinant Tul4 and FopA vaccine and the value of the hu-mice model for FT vaccine research. Overall, I suggest that such an approach might be widely applicable to vaccine studies of the FT.ABSTRACT i TABLE OF CONTENTS iv LIST OF FIGURES viii LIST OF TABLES x LIST OF ABBREVIATION xi GENERAL INTRODUCTION xiii LITERATURE REVIEW xvi CHAPTER I Synthetic Tul4 and FopA peptides cocktail of Francisella tularensis induced humoral and cell-mediated immunity in mice 1 1 INTRODUCTION 2 2 MATERIALS AND METHODS 4 2.1 Mice 4 2.2 Synthetic peptides and adjuvant preparation 4 2.3 Generation of bone marrow derived dendritic cells 4 2.4 In vitro DC stimulation assay 5 2.5 Mixed lymphocyte reaction (MLR) 6 2.6 Immunizations with peptides 6 2.7 Ex vivo spleen re-stimulation 6 2.8 Humoral and cellular immune response of the mice 7 2.9 Statistical analysis 8 3 RESULTS 9 3.1 Synthetic peptides-stimulated maturation of BMDCs 9 3.2 Allostimulatory function of BMDCs pulsed with synthetic peptides 11 3.3 Immune response of splenocytes in mice immunized with synthetic peptides 13 3.4 Antibody response in mice immunized with synthetic peptides 15 4 DISCUSSION 18 CHAPTER II Humanized mice for the evaluation of Francisella tularensis vaccine candidates 22 1 INTRODUCTION 23 2 MATERIALS AND METHODS 25 2.1 Production of recombinant Tul4 and FopA proteins 25 2.2 Mice 25 2.3 Establishment of humanized mice 26 2.4 Analysis of human cell engraftment 26 2.5 Vaccination of the humanized mice 26 2.6 Humoral immune response 27 2.7 Challenge test 28 2.8 Histopathological analysis 28 2.9 Statistical analysis 31 3 RESULTS 32 3.1 Cloning, expression, and purification of the rTul4 and rFopA 32 3.2 Human specific immune response after immunization in humanized mice 34 3.3 Partial protection against LVS in humanized mice immunized with rTul4 and rFopA 38 4 DISCUSSION 46 GENERAL CONCLUSION 49 REFERENCES 52 국문초록 73 Docto

인간 치주인대 줄기세포에 대한 최신의 칼슘 실리케이트 실러와 에폭시 레진 계 실러의 생체 적합성에 대한 비교 연구

The aim of this study was to evaluate various biocompatibility of newly introduced calcium silicate-based sealers (CeraSeal and EndoSeal TCS) and epoxy resin-based sealer (AH-Plus) on various aspect on human periodontal ligament stem cells (hPDLSCs). hPDLSCs were acquired from premolars (n = 4) of four subjects, whose ages extended from 16 to 24 years of age. To make media extracted from freshly mixed sealers (fresh media), all unset experimental sealers were mixed with culture medium (DMEM) and incubated for 24 h. To make media extracted from set sealers (setting media), materials were set in disc form for 48 h, and then extracted in DMEM for 24 h. hPDLSCs were cultured in fresh media or setting media according to each experimental condition. The expression of mesenchymal stem cell surface molecules (CD11b, CD19, CD34, CD45, CD73, CD90, CD105, and HLA-DR) was analyzed with flow cytometry (FACS) after culture in setting media. Cell viability was assessed using cell viability assay (Cell Counting Kit-8; CCK-8) after culture in fresh and setting media. To evaluate inflammatory response to the materials, concentrations of IL-6, IL-8, TGF-β in fresh and setting media were analyzed using ELISA kit. The osteogenic potential of hPDLSCs in setting media was quantified with RT-qPCR (Real Time-quantitative Polymerase Chain Reaction) for ALP, OCN, and RUNX2, and visually qualified with ALP (Alkaline Phosphatase) staining and ARS (Alizarin Red S) staining. Cell attachment on material and material surface morphology was evaluated with Scanning Electronic Microscopy. Statistical differences were assessed by analysis of variance followed by the Tukey’s test (p 99%) and the hematopoietic markers showed low expression level (<1%) in both Calcium silicate-based sealers and AH-Plus, therefore stemness of hPDLSCs was maintained in all materials. In cell viability test, AH-Plus showed the lowest cell viablity in all experimental periods, and CeraSeal showed significantly higher cell viability than others in fresh media (p < 0.05). In setting media, cell viablity was not significantly different between materials over all time periods. In ELISA test, AH-Plus showed higher expression of pro-inflammatory cytokines (IL-6 and IL-8) than other sealers. In the anti-inflammatory cytokine TGF-β, only CeraSeal maintained a similar level to control in setting media. In RT-qPCR test, AH-Plus showed lower expression level than other material, however EndoSeal TCS showed better expression level than others. As a result of ALP and ARS staining tests, calcium silicate-based sealers were stained similarly to positive control, and AH-Plus was less stained. Finally, scanning electron microscopy studies showed low degree of cell proliferation and differentiation on AH-Plus. However, CeraSeal and EndoSeal TCS showed high degree of cell proliferation and differentiation. Calcium silicate-based sealers are more biocompatible and less cytotoxic than epoxy-resin based sealer. In particular, CeraSeal showed less cytotoxicity than other materials before setting, and EndoSeal TCS showed better osteogenic potential than other materials.open석

Development of non-human primate models of human infectious disease for the Industry-University-Institute needs

국가 재난형 신·변종 감염병 발생 대비 맞춤형 감염병 모델 개발 및 산·학·연 활용 지원 사업KGM457201

Development of animal models with degenerative brain diseases

노인성 뇌질환 형질전환 동물모델 개발사업KGM462201

Establishment of customized drug efficacy assessment platform based on the comparative analysis of primate degenerative brain disease models

영장류 퇴행성 뇌질환 모델의 비교의학적 분석 데이터 기반 맞춤 약물 유효성 평가 플랫폼 구축KGM456201