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Abstrak :
[ABSTRAK
Demam berdarah dengue (DBD) merupakan penyakit yang disebabkan karena infeksi virus dengue (DENV), yang banyak ditemukan di Indonesia. Belum ada terapi yang spesifik dalam pengobatan DBD. Upaya pengembangan vaksin dengue yang efektif sangat diperlukan. Pada penelitian ini dilakukan analisa imunogenisitas kandidat vaksin DNA prM-E dengue serotipe 2 (pUMD2.kl.20) dengan menganalisis sel T CD4, sel T CD8, IFN-γ dan TNF-α. pada sel U937 dan PBMC secara in vitro, bertujuan untuk mengetahui respon imun ketika vaksin diinjeksikan ke dalam tubuh manusia. Dasar penelitian ini adalah sel U937 ditransfeksi dengan pUMD2.kl.20 menggunakan lipofectamin. Sel U937 akan berperan sebagai APCs yang akan mengekspresikan protein prM-E DENV-2 dan mempresentasikan protein tersebut melalui molekul MHC class I dan MHC class II kepada Peripheral Blood Mononuclear Cell (PBMC) manusia. Tahapan kerja yang dilakukan dalam penelitian ini terdiri dari: (a) kultur galur sel U937, (b) transfeksi sel U937 dengan pUMD2.kl.20, (c) transfeksi sel U937 dengan plasmid s(pUMVC), (d) infeksi sel U937 dengan DENV-2 strain DS.18/09, (e) pewarnaan dan pengamatan hasil pewarnaan menggunakan alat semi flowcytometri (TALI). pUMD2.kl.20 mengaktivasi sel T CD4 dan sel T CD8 untuk berploriferasi. Hasil penelitian ini menunjukkan bahwa konsentrasi sel T CD8 lebih tinggi dari konsentrasi sel T CD4 dan konsentrasi sel positif yang mensekresikan IFNγ lebih tinggi dari konsentrasi sel positif yang mensekresikan TNFα. Kondisi optimal dari aktivasi sel T CD4 oleh pUMD2.kl.20 adalah 24 jam setelah penambahan PBMC setelah transfeksi (8,53x10⁴sel/ml), untuk sel T CD8 adalah 24 jam setelah penambahan PBMC setelah transfeksi (49,4x10⁴ sel/ml). Kondisi optimal dari aktivasi sel+ yang mensekresikan IFNγ oleh pUMD2.kl.20 adalah 24 jam setelah penambahan PBMC 48 jam setelah transfeksi (9,86x10⁴ sel/ml), dan untuk sel+ yang mensekresikan TNFα adalah 2 jam setelah penambahan PBMC 48 jam setelah transfeksi (2,1x10⁴ sel/ml). Dari penelitian ini dapat disimpulkan bahwa pUMD2.kl.20 bersifat imunogenik.

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ABSTRACT
Dengue hemorrhagic fever (DHF) is a disease caused by infection with dengue virus (DENV), which is found in Indonesia. There is no specific therapy in the treatment of DHF. An effort to develop an effective dengue vaccine is needed. In this research, analysis of the immunogenicity of the DNA vaccine candidate prME dengue serotype 2 (pUMD2.kl.20) by analyzing the CD4 T cells, CD8 T cells, IFN-γ and TNF-α in U937 cells and PBMC in vitro, aims to determine the immune response when the vaccine is injected into the human body. This research approach is based on transfection of U937 cells with pUMD2.kl.20 using lipofectamin. U937 cells acts as APCs which will express the protein prM-E DENV-2 and presenting these proteins through the MHC class I and MHC class II molecule to the Peripheral Blood Mononuclear Cell (PBMC) of human body. Stages of the work in this study consisted of: (a) culture cell line U937, (b) transfection of U937 cells with pUMD2.kl.20, (c) transfection of U937 cells with plasmid (pUMVC), (d) infection of U937 cells with DENV-2 strains DS.18/09, (e) staining and observation results of staining using a semi-flow cytometri (TALI). pUMD2.kl.20 activate CD4 T cells and CD8 T cells to proliferate. CD8 T cells concentration higher than the concentration of CD4 T cells and the secretion of INFγ-positive cells concentration higher than the concentration of the secretion of TNFα-positive cells. Optimal condition of CD4 T cells activation by pUMD2.kl.20 is 24 hours after the addition of PBMC after transfection (8,53x10⁴ cells/ml), for CD8 T cells was 24 hours after the addition of PBMC after transfection (49,4x10⁴ cells/ml). Optimal conditions secretion of IFNγ-positive cells were activated by pUMD2.kl.20 is 24 hours after the addition of PBMC 48 hours after transfection (9,86x10⁴ cells/ml), and for the secretion of TNFα- positive cells were activated by pUMD2.kl.20 is 2 hours after the addition of PBMC 48 hours after transfection (2,1x10⁴ cells/ml). From this study it can be concluded that the pUMD2.kl.20 immunogenic, Dengue hemorrhagic fever (DHF) is a disease caused by infection with dengue virus (DENV), which is found in Indonesia. There is no specific therapy in the treatment of DHF. An effort to develop an effective dengue vaccine is needed. In this research, analysis of the immunogenicity of the DNA vaccine candidate prME dengue serotype 2 (pUMD2.kl.20) by analyzing the CD4 T cells, CD8 T cells, IFN-γ and TNF-α in U937 cells and PBMC in vitro, aims to determine the immune response when the vaccine is injected into the human body. This research approach is based on transfection of U937 cells with pUMD2.kl.20 using lipofectamin. U937 cells acts as APCs which will express the protein prM-E DENV-2 and presenting these proteins through the MHC class I and MHC class II molecule to the Peripheral Blood Mononuclear Cell (PBMC) of human body. Stages of the work in this study consisted of: (a) culture cell line U937, (b) transfection of U937 cells with pUMD2.kl.20, (c) transfection of U937 cells with plasmid (pUMVC), (d) infection of U937 cells with DENV-2 strains DS.18/09, (e) staining and observation results of staining using a semi-flow cytometri (TALI). pUMD2.kl.20 activate CD4 T cells and CD8 T cells to proliferate. CD8 T cells concentration higher than the concentration of CD4 T cells and the secretion of INFγ-positive cells concentration higher than the concentration of the secretion of TNFα-positive cells. Optimal condition of CD4 T cells activation by pUMD2.kl.20 is 24 hours after the addition of PBMC after transfection (8,53x10⁴ cells/ml), for CD8 T cells was 24 hours after the addition of PBMC after transfection (49,4x10⁴ cells/ml). Optimal conditions secretion of IFNγ-positive cells were activated by pUMD2.kl.20 is 24 hours after the addition of PBMC 48 hours after transfection (9,86x10⁴ cells/ml), and for the secretion of TNFα- positive cells were activated by pUMD2.kl.20 is 2 hours after the addition of PBMC 48 hours after transfection (2,1x10⁴ cells/ml). From this study it can be concluded that the pUMD2.kl.20 immunogenic]

Abstrak :
ABSTRAK
Vaksin baru sangat dibutuhkan untuk mengendalikan tuberkulosis TB . Protein yang disekresikan oleh M.tuberculosis diketahui dapat menginduksi kekebalan protektif. Pada genom M.tuberculosis terdapat protein yang berperan dalam reaktivasi M.tuberculosis, protein tersebut bernama resuscitation promoting factor Rpf . RpfD merupakan salah satu dari keluarga Rpf yang telah terbukti imunogenik sehingga membuat RpfD cocok untuk digunakan sebagai vaksin TB. Tujuan dari penelitian ini adalah untuk mengkonstruksi vaksin DNA yang menyandi gen rpfD dan menginvestigasi imunogenisitas vaksin tersebut pada mencit. Gen rpfD M.tuberculosis diamplifikasi menggunakan teknik PCR. Gen rpfD dan plasmid pcDNA3.1 kemudian dipotong dengan enzim restriksi EcoRI dan HindIII kemudian diligasi dan ditransformasikan ke E.coli DH5?. Plasmid rekombinan pcDNA3.1-rpfD yang telah diuji kebenaran urutan asam amino dan orientasinya ditransfeksikan ke dalam sel CHO-K1. Selanjutnya, mencit Balb/c diimunisasi setiap dua minggu sekali secara intramuskular dengan pcDNA3.1-rpfD. Antibodi spesifik yang terdapat di serum mencit dideteksi dengan Western Blot. ELISA digunakan untuk mengukur tingkat IL-12, IFN-?, IL-4, dan IL-10. Hasil penelitian ini menunjukkan bahwa telah terdeteksi antibodi yang spesifik terhadap pcDNA3.1-rpfD. Selain itu, vaksin DNA ini juga dapat menginduksi produksi IL-12 dan IFN-? tetapi tidak pada IL-4 dan IL-10.

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ABSTRACT
Novel vaccines are needed to control tuberculosis TB . Proteins secreted by M.tuberculosis are known to induce the protective immunity. In the M.tuberculosis genome, there is protein for which a possible role in reactivation of M.tuberculosis, this protein called resuscitation promoting factor Rpf . RpfD is one of the Rpfs family which proved to be immunogenic as make it suitable to be used as TB vaccine. The aim of this study was to construct the DNA vaccine encoding rpfD gene and to investigate its immunogenicity in mice. The rpfD gene of M.tuberculosis was amplified using PCR techniques. The rpfD gene and pcDNA3.1 plasmid are then digest with restriction enzymes EcoRI and HindIII then ligated and transformed to E.coli DH5 . The recombinant plasmid pcDNA3.1 rpfD that has been tested the sequences and its orientation is transfected into CHO K1 cells. Furthermore, Balb c mice were immunized every two weeks intramuscularly with pcDNA3.1 rpfD. The specific antibodies in the serum detected by Western Blot. ELISA was applied to determine the levels of IL 12, IFN , IL 4, and IL 10. The result showed that the specific antibody was detected in the serum mice. Besides that, DNA vaccine can induce IL 12 and IFN but not in IL 4 and IL 10 production.