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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 :

Demam berdarah dengue (DBD) merupakan penyakit infeksi global dengan insiden dan mortalitas tinggi. Virus dengue (DENV), penyebab dari infeksi ini, memiliki vektor nyamuk Aedes aegypti dan Aedes albopictus. Namun, hingga saat ini belum ada antivirus spesifik yang tersedia dan upaya pencegahan masih belum efektif. Propil galat merupakan unsur fenolik berpotensi sebagai kandidat antivirus DENV, tetapi belum diketahui bagaimana mekanisme penghambatannya. Pada penelitian ini, akan dilakukan analisis penghambatan reseptor sel dan pengikatan envelope DENV oleh propil galat untuk mengidentifikasi mekanisme propil galat sebagai antivirus DENV secara in vitro dan in silico. Studi ini merupakan eksperimen laboratorium untuk menganalisis mekanisme propil galat sebagai antivirus DENV pada tahap penempelan virus secara in vitro. Strain DENV yang digunakan adalah DENV-2 dan sel kultur yang digunakan adalah sel Vero. Persentase penghambatan diperoleh dengan metode focus assay, sedangkan persentase viabilitas menggunakan metode MTT assay. Selain itu, uji in silico dilakukan untuk mengetahui energi ikatan dan konstanta inhibisi propil galat terhadap protein E. Persentase penghambatan propil galat perlakuan reseptor dan protein E yakni berturut-turut sebesar 9 ± 2,65% dan 53 ± 9,85%. Persentase viabilitas pada perlakuan tahap penempelan virus ke sel adalah 125±1%. Energi ikatan propil galat terhadap protein E adalah -3,21 kkal/mol dengan konstanta inhibisi sebesar 4,44 mM. Propil galat memiliki efek penghambatan terhadap DENV-2 dan tidak toksik jika diberikan pada tahap penempelan virus secara in vitro. Energi ikatan antara protein E dengan propil galat sangat rendah sehingga ikatannya spontan dan afinitasnya baik. Propil galat lebih berpotensi sebagai profilaksis DENV-2.

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Dengue hemorrhagic fever (DHF) is a global infectious disease with a high incidence and mortality. Dengue virus (DENV), the etiologic agent, has Aedes aegypti and Aedes albopictus as its vectors. However, there is no specific antivirus available and prevention efforts are still ineffective. Propyl gallate is a potential candidate for DENV antivirus, but the mechanism is not yet known. In this study, inhibition of cell receptor and binding of DENV envelope by propyl gallate were conducted to identify its in vitro and in silico mechanism as an anti-DENV. This is an experimental study to analyze the mechanism of propyl gallate as  DENV antivirus in viral attachment stage. DENV-2 was the strain used and the culture cell used was the Vero cell. The inhibitory percentage was obtained by focus assay method, while the viability percentage using MTT assay method. In addition, in silico test was carried out to calculate propyl gallates binding energy and inhibition constant for protein E. The inhibitory percentage of propyl gallate towards receptor and viral envelope are 9±2.65% and 53±9.85%, respectively. The viability percentage in viral attachment inhibition was 125±1%. The binding energy of propyl gallate to protein E was -3.21 kcal/mol with inhibition constant of 4.44 mM. Hence, propyl gallate has an inhibitory effect on DENV-2 and is non-toxic if given in viral attachment stage. The low binding energy between protein E and propyl gallate shows spontaneous bond with a good affinity. Propyl gallate is more potential as DENV-2 prophylaxis.

 

Abstrak :
ABSTRAK Infeksi yang disebabkan oleh virus dengue telah banyak dilaporkan di negara tropis dan subtropis. Virus dengue terdiri dari 4 serotipe yaitu dengue 1-4. Hingga saat ini belum tersedia vaksin yang berlisensi untuk mencegah terjadinya infeksi dengue. Pada penelitian ini dikonstruksi vaksin DNA yang mengkode gen prM-E dan prM-E-NS1del virus dengue 2 strain Indonesia yang akan dijadikan sebagai kandidat vaksin dengue. Hasil penelitian berhasil mendapatkan 9 plasmid rekombinan pUMDE2 yang membawa gen sisipan prM-E dan telah dikonfirmasi dengan melakukan PCR koloni dan restriksi plasmid. Dari hasil sekuensing plasmid pUMDE2 koloni no. 11 ditemukan 19 mutasi asam amino pada gen prM-E, sepuluh mutasi pada gen prM dan sembilan mutasi pada gen E. Mutasi protein prM N29D dan N52K serta protein E V164I dan S390N terletak pada daerah epitop pengenalan sel B. Transfeksi plasmid pUMDE2 dilakukan pada sel Chinese Hamster Ovary (CHO)-K1 dan menunjukkan adanya ekspresi protein prM-E rekombinan berdasarkan uji imunostaining dan ELISA. Hasil ELISA menunjukkan bahwa protein ditemukan pada sel yang ditransfeksi. Sedangkan, plasmid rekombinan yang membawa gen prM-E-NS1del tidak berhasil dikonstruksi. Plasmid pUMDE2 dapat dikembangkan menjadi kandidat vaksin DNA.

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ABSTRACT Infection by dengue virus were reported in tropical and subtropical area. Dengue virus (DENV) consist of 4 serotype, DENV-1 to DENV-4. There is no licensed vaccine available for dengue infection. In this research, we construct DNA vaccine encode prM-E and prM-E-NS1del genes of dengue virus serotype 2 for vaccine development. Nine recombinant plasmids that encode prM-E genes (pUMDE2), were successfully obtained. Recombinant plasmids were confirmed by PCR colony and restriction enzyme analysis. Colony of pUMDE2 no. 11 was sequenced and total 19 amino acid mutations were founds, 10 mutations in prM and 9 mutations in E protein. prM mutations N29D and N52K, E mutations of V164I and S390N were found in B cell epitopes. Transfection pUMDE2 plasmid was done to Chineese Hamster Ovary (CHO)-K1 and showed that recombinant protein prM-E was successfully expressed by immunostaining assay and ELISA. Results showed that the protein was mainly found in cell fraction. However, recombinant plasmid that encode prM-E-NS1del were failed to be constructed. pUMDE2 could be developed for vaccine candidate.