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G protein fragment derived from Respiratory Syncytial Virus A2 as a vaccine candidate in mice administered via sublingual route : 호흡기성 융합 바이러스 A2에서 유래된 G 단백질 조각을 이용한 설하 접종 백신 후보 물질 연구

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dc.contributor.advisor윤철희-
dc.contributor.author천인수-
dc.date.accessioned2017-07-13T08:20:09Z-
dc.date.available2017-07-13T08:20:09Z-
dc.date.issued2014-02-
dc.identifier.other000000017515-
dc.identifier.urihttps://hdl.handle.net/10371/119456-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 윤철희.-
dc.description.abstractRespiratory syncytial virus (RSV) is one of the major pathogens causing respiratory tract infection and disease in infants, young children and immunocompromised patients worldwide. In addition, almost all children have experienced RSV infection by 2 to 3 years of age. However, currently there is no vaccine available for human use.
RSV G glycoprotein (RSVG) plays a role in attachment on the host cell and induces neutralizing antibody response which is involved in protective response against RSV infection in the host. However, RSVG, especially CD4+ T cell epitope region within RSV G protein, is known to induce immunopathology.
Mucosal vaccination could induce humoral and cellular immune responses at systemic compartment and mucosal site. However, intranasal route is known to redirect antigen to central nerve system that leads to serious side effect like Bells palsy although it is rare. In contrast, sublingual route is seemingly safer than intranasal route despite the fact that its immunogenicity is low. The purpose of this study is to examine RSV vaccine candidate without immunopathology using RSV G protein core fragment (Gcf) administered through sublingual route in mouse.
In the first study, the effect of Gcf immunization routes via sublingual or intranasal routes in mouse was compared when RSV was infected subsequently. Unexpectedly, Gcf sublingual vaccination induced vaccine-enhanced disease, mediated by Th17 response, but intranasal vaccination of Gcf did not vaccine-enhanced disease. The vaccine-enhanced disease by formalin inactivated-RSV (FI-RSV) was mediated through Th2 response as it has been already reported.
In the second study, since CD4+ T cell epitope within RSV G protein was reported to be responsible for the immune response and immunopathology, it was modified for safe and effective RSV vaccine when administered by sublingual route. The amino acid (a.a.) positions 185 and 188, critical sites to activate T cells, were mutated to generate mGcf in order to vanish vaccine-enhanced disease. Then, to recover the function of CD4 helper T cell activity, mGcf was fused with CD4+ T cell epitope from RSV F (F51-66) protein, and generated Th-mGcf. As a result, Th-mGcf induced humoral immune response and provided protection against RSV without immunopathology.
Taken together, vaccine-enhanced disease induced in mice sublingually immunized with Gcf following RSV challenge was mediated by Th17 response. The mutation of CD4+ T cell epitope in Gcf (mGcf) resulted in lower immunogenicity without immunopathology. However, lower immunogenicity by mutation of CD4+ T cell epitope was recovered by conjugation of another CD4+ T cell epitope from RSV F protein (Th-mGcf). Finally, this Th-mGcf immunization via sublingual route after RSV challenge induced protective immune response without vaccine enhanced disease and it might be potential candidate for RSV vaccine.		-
dc.description.tableofcontentsAbstract I
Contents IV
List of Figures IX
List of Tables XI
List of Abbreviations XII
Review of Literature 1
1. RSV 1
1.1 Characteristics 1
1.2 Epidemiology 4
1.3 Immune responses and pathogenesis in RSV infection 5
1.3.1 Immune responses 5
1.3.2 Immunopathogenesis 8
1.3.3 Viral evasion 10
1.4 Animal models and limitation 11
1.4.1 Mice 11
1.4.2 Cotton rats 12
1.3.3 Non-human primate 12
2. RSV vaccine 13
2.1 Types of RSV vaccine 13
2.1.1 FI-RSV 13
2.1.2 Live attenuated RSV vaccine 16
2.1.3 Vector-based RSV vaccines 18
2.1.4 Subunit vaccines 19
2.2 Vaccine-enhanced pathogenesis 20
2.3 Factors for effective RSV vaccine 24
3. RSV G glycoprotein 27
3.1 Characteristics 27
3.2 T cell epitope and its modification 28
3.3 RSV G glycoprotein as an RSV vaccine candidate 30
4. References 32
General Introduction 60
Chapter 1. Comparison of vaccine-enhanced disease by sublingual and intranasal routes in mice immunized with G protein fragment derived from Respiratory Syncytial Virus A2 following viral infection 63
1. Introduction 64
2. Materials and Methods 66
1) Materials 66
2) Virus preparation 66
3) Construction of plasmids and purification of RSV G protein fragment 66
4) Mice and immunization 67
5) Cytokines 68
6) T cell response 69
7) Measurement of eosinophils or neutrophils in the BAL or lung 69
8) ELISA for detection of antibodies 70
9) Virus titration in mouse lung 71
10) Histology 71
11) Statistical analysis 71
3. Results 73
1) Effect of administration route on the immune response and pathogenesis following RSV infection 73
2) Histological effect by G protein fragment administration route 77
3) Inflammatory cytokines induced by G protein fragment vaccination route following RSV infection 80
4) T cell response by G protein fragment vaccination route 82
5) Infiltration of granulocytes 90
6) Effect of IL-17 on vaccine-enhaced immunopathology 97
4. Discussion 99
5. References 103
Chapter 2. Development of safe and effective RSV vaccine by modified CD4 epitope in G protein core fragment 109
1. Introduction 110
2. Materials and Methods 113
1) Ethics statement 113
2) Virus preparation 113
3) Construction of plasmids expressing various Gcf proteins 113
4) Expression and purification of various Gcf proteins 116
5) Mice and immunization 117
6) T cell response 117
7) Measurement of eosinophils in BAL 118
8) ELISA 119
9) Virus titration in mouse lung 119
10) Statistical analysis 120
3. Results 121
1) Effect of CD4+ T cell epitope in wtAGcf on the immune response and pathogenesis following RSV A subtype infection 121
2) Immune response and eosinophilia by wtBGcf after RSV B subtype infection 125
3) Amino acid residues 183-195 within RSV BGcf lack functionality as CD4+ T cell epitope 129
4) T cell response by Gcfs 131
5) Immunogenecity and protective efficacy of recombinant Gcfs against homologous RSV A type infection 133
6) Immunogenecity and protective efficacy of recombinant Gcfs against heterologous RSV B subtype infection 137
4. Discussion 141
5. References 145
General Conclusion 150
Summary in Korean 152		-
dc.formatapplication/pdf-
dc.format.extent3614309 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectRespiratory Syncytial Virus-
dc.subject.ddc630-
dc.titleG protein fragment derived from Respiratory Syncytial Virus A2 as a vaccine candidate in mice administered via sublingual route-
dc.title.alternative호흡기성 융합 바이러스 A2에서 유래된 G 단백질 조각을 이용한 설하 접종 백신 후보 물질 연구-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesXIV, 154-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2014-02-
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