Studies on DNA-based vaccines of Japanese encephalitis : 일본뇌염 DNA 백신에 관한 연구

다국어 초록 (Multilingual Abstract)

It was analysed nucleotide and amino acid sequences of JEV protective genes, and investigated the immunogenicity of plasmid DNA expressing NS1 (nonstructural protein 1) protein, prM (premembrane) and E (envelope) in mice as an animal model. The nucleotide sequence of pr (prM signal sequence), M, E and NS1 gene of attenuated JEV Anyang strain which have been used as a vaccine strain for veterinary use in Korea was determined. The nucleotide sequences of Anyang strain were the highest homology with JaOArS982 strain of 18 JEV strains. The nucleotide and amino acid sequences from pr, M, E and NS1 regions between Anyang strain and JaOAr982 strain were 98.8 and 98.9%, respectively. The homologies of nucleotide and amino acid sequence of E region between Anyang strain and JaOAr982 strain were 98.9 and 98.8%, respectively. The phylogenetic analysis shows that JaOArS982 strain is very close to Anyang strain. No overlapping amino acid residues were found in the pr, M, E NS1 protein sequences among eight attenuated variant JEV strains; P3MRPvar, JE-B, Nak-rec, CH2195LA, RP-2ms, SA-14-14-2, SA-14-2-8 and SA-14-5-3. This suggests that the JEV virulence phenotype would be influenced by the strain-specific virulence determinants. The phylogenetic relationship in subgroups indicated that the attenuated Anyang strain was not similar to any of the eight attenuated strains.
To induced immune responses to prME and NS1 proteins of JEV, the genes were constructed to express prME and NS1 proteins under control of the human cytomegalovirus (HCMV) immediate-early promoter and intron A. After DNA transfection into COS-7 cells to check the prME expression level, the hemagglutination (HA) test for E containing HA domain was performed. HA titer in supernatant was higher than that in cell lysates. The expressions of prME and NS1 gene of JEV inserted into DNA vaccine vector, pSLIA, were identified by immuoprecipitation with anti-NS1 and E monoclonal antibodies. The molecular weights of NS1 and E proteins immunoprecipitated with specific antibodies expressed in pSLIA were approximately 46 Kda and 54 Kda, respectively.
The HI titers were 1:40 in the mice immunized with 10 pg of pSLIA-prME, and 1:10 in the mice immunized with each 10 pg of combined pSLIA-NS1 and pSLIA-prME at 2 weeks after the third immunization by intradermal (ID) routes. The HI titers were 1:20 in the mice immunized with 100 pg of pSLIA-prME by intramuscular (IM) route and <1:10 in combined pSLIA-NS1 and pSLIA-prME by IM route. Co-administration of pSLIA-NS1 and pSLIA-prME did not have a significant enhancement effect on the total serum antibody titers. HI antibodies in the group by IM route was not higher than those in the groups by IM route. The virus neutralization (VN) titers after the third immunization were <1:2 to 1:2 in the mice immunized with pSLIA-prME at the back by ID route, <1:2 to 1:32 in the mice immunized with pSLIA-prME at the quadricep muscles by IM route, and 1:4 to 1:16 in the mice immunized with pSLIA-prME at the tail by ID route.
After immunizations with pSLIA-prME, the predominant isotype in the mice immunized by IM route was IgG2a and the mice immunized by ID route showed also predominantly IgG1 type antibody response. After co-administration with pSLIA-prME and pSLIA-NS1, the predominant isotype of anti-prME antibody in the mice immunized by IM route was IgG2a and the mice immunized by ID route showed also predominantly IgG1 type antibody response. After immunization with pSLIA-NS1, the predominant isotype in the mice immunized by IM route was IgG1 and IgG2b, and the mice immunized by ID route showed also predominantly IgG1 and IgG2b type antibody response. The antibodies of IgG2a were almost not shown in the group immunized with pSLIA-NS1 or combined pSLIA-NS1 and pSLIA-prME, and by ID or IM routes, either.
The reactivity of IFN-y secreting cells stimulated with NS1 antigen showed the significant results that immunization with combined pSLIA-prME and pSLIA-NS1 induced more secretion of IFN- z than immunization with pSLIA-NS1. Reactivity for IFN- r secreting cell to prME in the mice immunized by only IM route showed the significant results when compared with control.
Effects of the plasmid expressing prME and NS1 proteins on the cytokine profiles in spleens and IgG isotypes in the serum from the immunized mice were examined by the secreted cytokines of IFN- y and IL-4, and IgG subclasses of IgG1 and IgG2a. The results show that Thl response increased in the group immunized with pSLIA-prME by IM route, while Th2 response increased in the group immunized with pSLIA-prME by ID route.
The lymphocyte proliferation from the mice immunized with pSLIA-NS1, combined pSLIA-prME and pSLIA-NS1 by ID route also were stronger than that from the mice with pSLIA, and the proliferation response of the mice immunized with pSLIA-prME or combined pSLIA-prME and pSLIA-NS1 by ID route showed also significant increase but the mice with pSLIA-prME or combined pSLIA-prME and pSLIA-NS1 by IM route showed not significant increase when compared with control. To enhance the immune response, the various immunized routes and sites were examined. The significant levels of anti-prME antibodies after 1st immunization at tail by ID route were detected. However, the specific antibody for anti-prME in the mice immunized at back by ID and at quadriceps muscle by IM route was not detected at the primary immunization. The results indicated that the sites of immunization affect the efficacy of immunity of DNA-based vaccines.
The prME and NS1-based DNA vaccines did elicit both humoral and cellular immunity for Japanese encephalitis in mouse. These results suggest that DNA-based vaccine may be a candidate of preventive measure for Japanese encephalitis virus infection.

목차 (Table of Contents)

• 목차
• List of Figures = v
• List of Tables = vii
• List of Abbreviations = viii
• I. Introduction = 1
• 1.1. Japanese encephalitis virus = 1
• 1.2. Relationships of JEV with other viruses = 2
• 1.3. Physicochemical properties = 3
• 1.4. Structure of viral genome = 3
• 1.5. Feature of the viral proteins = 5
• 1.5.1. Capsid = 5
• 1.5.2. prM and M = 5
• 1.5.3. Envelope (E) = 5
• 1.5.4. NS1 = 6
• 1.5.5. NS2 = 7
• 1.5.6. NS3 = 7
• 1.5.7. NS4 = 7
• 1.5.8. NS5 = 8
• 1.6. Virus cultivation = 8
• 1.7. Transmission cycles of virus = 9
• 1.8. Serological studies = 10
• 1.9. NuceIotide sequence analysis = 11
• 1.10. Molecular epidemiological analysis = 12
• 1.11. Pathology = 16
• 1.11.1. Clinical signs = 16
• 1.11.2. Pathogenesis = 17
• 1.11.3. Immunity of JEV infection = 18
• 1.11.4. Lesions = 19
• 1.12. Diagnosis = 21
• 1.13 Prevention = 22
• 1.13.1. Vaccine = 22
• 1.13.2. Trend of vaccine development = 22
• 1.13.3. Use of vaccine = 23
• 1.14. Expression of JE recombinant proteins = 24
• 1.15. JE studies in Korea = 26
• 1.15.1 Occurrence and virus isolation = 26
• 1.15.2. Epidemiology = 26
• 1.15.3. Development of diagnostic methods and vaccines = 27
• 1.16. Development of DNA vaccine = 27
• 1.16.1. History of DNA vaccine = 27
• 1.16.2. Mechanism of DNA vaccine = 28
• 1.16.3. Advantage of DNA vaccine = 30
• 1.17. Regulation of immunity by cytokines = 33
• 1.18 Aims of studies = 34
• II. Materials and Methods = 36
• 2.1 Viruses and cells = 36
• 2.2. RNA isolation and reverse transcription- polymerase chain reaction (RT-PCR) = 36
• 2.3. NucIeotide sequencing = 37
• 2.4. Phylogenetic analysis = 37
• 2.5. Construction of plasmid expressing prM, E and NS1 protein of JEV = 43
• 2.6. Preparation of plasmid for DNA vaccine = 43
• 2.7. Transfertion into cells = 44
• 2.8. PCR identification of cloning = 45
• 2.9. Production of anti-JEV monoclonal antibodies = 45
• 2.10. Recombinant proteins expressed by baculovirus containing NS1 gene = 46
• 2.11. Immunoprecipitation = 46
• 2.12. Immunoprecipitation of DNA vaccine = 47
• 2.13. Hemagglutination (HA) test of recombinant proteins = 47
• 2.14. Hemagglutination inhibition (HT) test = 52
• 2.15. Preparation of antigen for lymphocyte stimulation or ELISA = 52
• 2.16. Anti-NS1 or anti-prME antibody detection by ELISA = 53
• 2.17. IgG subclass typing of anti-prME or anti-NS1 antibody by ELISA = 54
• 2.18. Virus titration and virus neutralizing test = 54
• 2.19. Immunofluorescence assay = 55
• 2.20. ELISPOT assays for IFN-γ and IL-4 secreting cells = 56
• 2.21. Cell proliferation assays = 56
• 2.22. Statistical analyses = 57
• III. Results = 58
• 3.1. Nucleotide and amino acid sequence of prM, E, NS1 gene of JEV = 58
• 3.2. Homology comparisons of nucleotide and amino acid sequences = 59
• 3.3. Major difference of deduced amino acids of JEV strains = 61
• 3.4. Phylogenetic relationships of JEV strains = 62
• 3.5. Anti-JEV Mabs and NS1 proteins expressed in Sf-9 cells by baculovirus = 69
• 3.6. Construction of plasmid vectors expressing JEV strains = 69
• 3.7. Identification of expressed proteins by immunoprecipitation and immunofluorescence = 75
• 3.8. Expression efficiency in pSLIA-prME transfected COS-7 cells = 75
• 3.9. Immunogenicity of DNA vaccines in mice = 84
• 3.10. The HI antibody in the mice immunized with DNA-based vaccine = 84
• 3.11. Virus neutralization in the mice immunized with DNA-based vaccine = 85
• 3.12. Anti-prME or anti-NS1 antibody detection by ELISA = 86
• 3.13. IgG subclass of anti-NS1 or anti-prME antibody = 87
• 3.14. Frequency of IEN-γ secreting cells in the mice spleens stimulated by NS1 antigen = 88
• 3.15. Frequency of IEN-γ secreting cells in spleens stimulated by prME antigen = 89
• 3.16. Frequency of IL-4 secreting cells in spleens stimulated by NS1 or prME antigen = 90
• 3.17. Cell proliferation in spleens stimulated by NS1 antigen = 91
• 3.18. Cell proliferation in spleen cells stimulated by prME antigen = 92
• 3.19. Immune enhancement in the mice immunized bv various sites or routes = 93
• 3.20. Type of immune response = 94
• IV. Discussion = 113
• 4.1. Comparison of nucleotide or amino acid sequences = 113
• 4.2. Phylogenetic relationships of JEV strains = 116
• 4.3. pSLIA as a vector for DNA-based vaccines = 118
• 4.4. prME or NS1 proteins expressed in COS-7 cells using pSLIA = 120
• 4.5. Humoral immunity = 122
• 4.6. Cellular imnaunity = 124
• 4.7. T helper response = 126
• 4.8. Enhancement of immune response = 130
• V. Conclusion = 132
• VI. References = 135
• ABSTRACT = 169
• SUMMARY IN KOREAN = 174