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The Efficacy of DNA Vaccine Using a Sindbis Virus-based Vector Against Nucleocapsid Protein of Hantaan Virus

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dc.contributor.advisor김, 완기-
dc.contributor.author우, 규진-
dc.date.accessioned2011-02-08T07:30:43Z-
dc.date.available2011-02-08T07:30:43Z-
dc.date.issued2006-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/1447-
dc.description.abstract"Hantaan virus (HTNV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). More than 100,000 cases of HFRS are reported yearly, with a mortality rate of between 2% and 10%. But, there is no effective and safe vaccine against HFRS. Even though neutralizing antibodies against the HTNV have been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. This study has examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination to investigate the immunogenicity of NP.

To examine the cellular immune response against HTNV NP, C57BL/6 mice were injected with HTNV intraperitoneal. The NP-specific CD8+ T cell response was analyzed using a 51Cr-release assay, intracellular cytokine assay, enzyme-linked immunospot assay and tetramer binding assay against H-2Kb restricted CTL epitopes of NP (M6 and N1 peptide). Using these methods, it was found that HTNV infection elicited a strong NP-specific CD8+ T cell response at 8 days after infection, and several different methods to check the NP-specific CD8+ T cell response showed a perfect correlation among analyses. To examine the humoral immune response against HTNV NP, the NP-specific antibody response was analyzed using an enzyme linked immunosorbent assay (ELISA). HTNV infection elicited the NP-specific humoral immune response which was began at 4~5 days after infection.

DNA vaccine has been shown to elicit both humoral and cellular immune responses, and confer protection against some viral, bacterial and parasitic pathogens. Therefore, DNA vaccine strategy was applied to HTNV in this study. Sindbis virus-based expression vector was carefully designed and constructed, in order to induce the transient high level expression of target gene. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, a single dose injection of 100㎍ of plasmid DNA into quardriceps muscle of C57BL/6 mice induced a high level of humoral and cellular immune response. The NP-specific antibody response was elicited 2~4 weeks after immunization and maximized at 6~10 weeks and sustained for over 14 weeks. NP-specific CD8+ T cell response reached its peak 2~3 weeks after immunization. Even though NP-specific CD8+ T cell response after DNA vaccination was not strong as the HTNV infection, but the pattern of response was similar to that of HTNV infection.

In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100 fold compared control mice. Even though challenge with rVV-HTNV-N in HTNV infected mice were perfectly protected, but DNA vaccination showed the partial protection.

In conclusion, this study showed that (i) HTNV infection in C57BL/6 mice elicited the strong NP-specific CD8+ T cell response at 8 days after infection, (ii) DNA vaccination with plasmid encoding HTNV nucleocapsid gene also elicited the strong NP-specific humoral and cellular immune responses, and (iii) DNA vaccination elicited the partial protective immunity against challenge with the recombinant vaccinia virus expressing NP. (iv) DNA vaccine expressing HTNV NP was shown to be a possible vaccine candidate against HTNV infection.

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dc.description.tableofcontents"TABLE OF CONTENTS



ABSTRACT ----------------------------------------------------------------------- i

TABLE OF CONTENTS -------------------------------------------------------- iv

LIST OF FIGURES -------------------------------------------------------------- vii

LIST OF TABLES --------------------------------------------------------------- ix

ABBREVIATION ---------------------------------------------------------------- x

I. INTRODUCTION ------------------------------------------------------------- 1

A. Immune responses against virus infection ------------------------------ 1

B. Vaccine development strategy and DNA vaccine ---------------------- 3

C. Enhancing the antigen expression ---------------------------------------- 7

D. Sindbis virus vector -------------------------------------------------------- 14

E. Hantaan virus --------------------------------------------------------------- 16

F. Objectives of this study ---------------------------------------------------- 19

II. MATERIALS AND METHODS ------------------------------------------- 20

A. Plasmid DNA and reagents ------------------------------------------------ 20

B. Mice, virus and cells -------------------------------------------------------- 20

C. Cloning of HTNV nucleocapsid gene and identification of

nucleocapsid protein --------------------------------------------------------- 21

1. PCR amplification and sequence analysis --------------------------- 21

2. In vitro transcription and translation (TNT) ------------------------- 22

D. Construction of DNA vaccine vector ------------------------------------ 23

E. Construction of DNA vaccine encoding HTNV nucleocapsid gene --- 26

F. Establishment of the assay systems for immune responses against NP-- 26

1. Assay for humoral immune response --------------------------------- 26

2. Assay for cellular immune response ---------------------------------- 29

G. In vivo test -------------------------------------------------------------------- 32

1. Infection of mice with live HTNV ------------------------------------ 32

2. Immunization of mice with DNA vaccines -------------------------- 34

H. Generation of recombinant vaccinia virus expressing HTNV NP

(rVV-HTNV-N) -------------------------------------------------------------- 35

1. Generation of rVV-HTNV-N ------------------------------------------ 35

2. Challenge studies using the recombinant vaccinia virus ----------- 39

III. RESULTS --------------------------------------------------------------------- 40

A. Cloning of HTNV nucleocapsid gene and identification of

nucleocapsid protein ------------------------------------------------------- 40

B. Construction of DNA vaccine vectors ----------------------------------- 40

C. Construction of DNA vaccine vectors expressing HTNV NP -------- 45

D. Establishment of the assay system for in vivo test --------------------- 45

1. Assay of humoral immune response --------------------------------- 47

2. Assay of cellular immune response ---------------------------------- 53

E. In vivo test ------------------------------------------------------------------- 59

1. Immune responses after HTNV infection --------------------------- 59

2. Immune responses after DNA vaccination -------------------------- 64

F. Generation of recombinant vaccinia virus expressing HTNV NP

(rVV-HTNV-N) ---------------------------------------------------------- 72

1. Selection and screening of recombinant virus plaques ---------- 72

2. Challenge studies using recombinant vaccinia virus ------------ 72

IV. DISCUSSION -------------------------------------------------------------- 77

V. CONCLUSION ------------------------------------------------------------- 83

REFERENCES ----------------------------------------------------------------- 85

국문요약 ------------------------------------------------------------------------ 107		-
dc.description.tableofcontentsLIST OF FIGURES



Fig. 1. Factors influencing efficacy of DNA vaccines. ---------------------- 12

Fig. 2. Construction of sindbis virus-based DNA vaccine vectors. -------- 25

Fig. 3. Schematic presentation of the tetramer analysis to detect CD8+

T lymphocytes. ----------------------------------------------------------- 33

Fig. 4. Construction of a vaccinia transfer plasmid for the generation

of recombinant vaccinia virus. ------------------------------------------- 37

Fig. 5. Cloning of HTNV nucleocapsid gene. --------------------------------- 41

Fig. 6. Comparison of marker gene expression among the constructed

sindbis virus-based DNA vaccine vectors. --------------------------- 44

Fig. 7. Construction of plasmid encoding nucleocapsid gene and

confirmation of NP expression in vitro. -------------------------------- 46

Fig. 8. Selection of recombinant baculovirus expressing HTNV NP. ----- 48

Fig. 9. Analysis of subcellular localization of NP expressed in insect cells. - 49

Fig. 10. Purification of recombinant NP expressed by baculovirus

expression system. ------------------------------------------------------- 50

Fig. 11. Establishment anti-HTNV NP ELISA conditions. -------------------- 52

Fig. 12. Screening of transformed E. coli that expressed H2-kb heavy

chain and mouse β2M.. ------------------------------------------------------- 54

Fig. 13. Preparation of inclusion body in E. coli. ------------------------------- 55

Fig. 14. Refolding and biotinylation of purified heavy chain and β2M. -------- 56

Fig. 15. Generation of tetramer by mixing with biotinylated MHC complex

and PE-conjugated streptavidin. ------------------------------------------ 58

Fig. 16. Immune responses in HTNV infected C57BL/6 mice. --------------- 62

Fig. 17. Comparison of antibody responses by DNA vaccine injection site. -- 65

Fig. 18. Comparison of antibody responses between pcDNA3-N

and CSHAC-N by injection dose. ---------------------------------------- 66

Fig. 19. Cellular immune response after DNA vaccination in C57BL/6 mice. - 68

Fig. 20. Humoral immune response after DNA vaccination in C57BL/6 mice.- 71

Fig. 21. Isolation and confirmation of recombinant vaccinia virus expressing

HTNV NP. ------------------------------------------------------------------- 74

Fig. 22. Protective immune response of HTNV NP expressing DNA vaccine.- 75

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dc.language.isoen-
dc.titleThe Efficacy of DNA Vaccine Using a Sindbis Virus-based Vector Against Nucleocapsid Protein of Hantaan Virus-
dc.title.alternativeSindbis virus-based vector를 이용한 한탄바이러스 Nucleocapsid 단백질에 대한 DNA vaccine의 효과-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001017-
dc.subject.keywordHantaan virus-
dc.subject.keywordDNA vaccine-
dc.description.degreeDoctor-
dc.contributor.department대학원 의학과-
dc.contributor.affiliatedAuthor우, 규진-
dc.date.awarded2006-
dc.type.localTheses-
dc.citation.date2006-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
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