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"[ABSTRAKPendahuluan: Infeksi dengue merupakan salah satu penyakit endemik di daerah tropis dan subtropis yang disebabkan oleh virus dengue (DENV). Hingga saat ini belum ada antiviral yang efektif untuk infeksi dengue. Penyebaran dan sirkulasi serotipe DENV berfariasi di setiap lokasi geografi, hal ini menyulitkan dalam melakukan evaluasi vaksin DENV. Oleh karena itu perlu dikembangkan kandidat vaksin DENV menggunakan strain Indonesia supaya dapat memberikan proteksi maksimal. Pada peneltian ini dikembangkan kandidat vaksin DNA tetravalen DENV berbasis gen prM-E DENV strain Indonesia.Metode: Konstruksi plasmid rekombinan kandidat vaksi dilakukan dengan cara menyisipkan gen prM-E setiap serotipe DENV ke dalam vektor pUMVC4a. Gen prM-E DENV merupakan strain Indonesia, yang diamplifikasi dari serum pasien yang terinfeksi dengan virus ini. Kemampuan plasmid rekombinan mengekspresikan protein prM-E DENV diuji di sel mamalia. Kemampuan kandidat vaksin menginduksi respon imun humoral dievaluasi secara monovalen dan tetravalen di mencit jenis ddY. Titer IgG anti dengue diperiksa menggunakan teknik ELISA, sedangkan titer antibodi netralisasi di tentukan dengan uji FRNT. Proteksi vaksin terhadap mencit yang diimunisasi dievaluasi dengan melakukan uji tantang menggunakan sel K562 yang diinfeksi DENV-2. Viremi virus di tentukan dengan menggunakan teknik foccus assay.Hasil: Konstruksi plasmid rekombinan kandidat vaksin DENV-1 dan DENV-3 sudah berhasil dilakukan. Plasmid dapat mengekspresikan protein prM-E DENV di sel mamalia, namun karakteristik dan kinetik protein masih belum dapat diketahui dengan jelas. Keempat kandidat vaksin DNA yang sedang dikembangkan dapat menginduksi respon imun, baik secara monovalen maupun tetravalen. Imunisasi secara tetravalen dapat memberikan proteksi pada mencit yang diuji tantang dengan sel K562 yang diinfeksi dengan DENV-2.;

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ABSTRACTIntroduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.;Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2., Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate.Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay.Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.]"

"ABSTRAKVirus dengue DENV dapat menginfeksi manusia tanpa batasan usia di daerah tropis dan subtropis. Vaksin DENV dari keempat serotype sangat diperlukan untuk mencegah infeksi DENV. Tujuan dari penelitian ini untuk melihat respon imun seluler CD4, CD8, dan CD25 pada mencit yang diimunisasi dengan vaksin DNA pUMD4 kla/b. Plasmid pUMD4 kla/b diproduksi dan diisolasi dengan menggunakan berbagai metode. Uji ekspresi pUMD4 kla/b dilakukan dengan transfeksi pada sel Chinese Hamster Ovary. Plasmid yang telah mengekspresikan protein preM-E DENV-4 selanjutnya diimunisasikan pada mencit ddY pada hari ke-0, ke-21, dan ke-42. Hasil analisis limpa tanpa induksi dengan menggunakan uji flow cytometry menunjukkan persentase CD4 pada mencit yang diimunisasi lebih rendah jika dibandingkan dengan kelompok pUMVC4a dan kelompok tanpa imunisasi. Akan tetapi persentase CD8 dan CD25 menunjukkan hasil yang lebih tinggi jika dibandingkan dengan kelompok pUMVC4a dan kelompok tanpa imunisasi. Analisis limpa dengan induksi pada mencit yang diimunisasi sebesar 3,7 CD4 , 9,7 CD8 , dan 13 CD25 secara berurutan dan persentase CD4, CD8, dan CD25 lebih tinggi jika dibandingkan dengan kelompok pUMVC4a dan kelompok tanpa imunisasi setelah imunisasi ke-3. Kesimpulan penelitian ini adalah adanya aktivasi imun seluler pada mencit setelah imunisasi dengan pUMD4 kla/b.

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ABSTRACTDengue virus infected humans in every ranges of ages at tropical and subtropical regions. In previous study DNA vaccine pUMD4 kla b was constructed. The purpose of this research is to inform cellular immune responses CD4, CD8, and CD25 in mice those were immunised by pUMD4 kla b. pUMD4 kla b plasmid was isolated by many methods. Expression test of pUMD4 kla b was held by transfection on CHO cells. pUMD4 kla b that had expressed preM E dengue proteins was immunised in ddY mice in aged 5 6 weeks on day 0, day 21, and day 42. Evaluation of immunizations could be seen from flow cytometry test on mice rsquo s splenocytes. pUMD4 kla b could express preM E dengue proteins. Result showed enhancements on percentages rsquo numbers of CD4 cells 2.6 , CD8 cells 4.4 , and CD25 6 in ddY mice without induction, and CD4 cells 3.7 , CD8 cells 9.7 , and CD25 13 with induction after third immunizations. Percentages of CD4, CD8, and CD25 in pUMD4 kla b rsquo s immunizations are higher than in pUMVC4a rsquo s immunizations and without immunizations. Conclusion there were cellular immunity activations after immunized with pUMD4 kla b."

"DNA vaccine is a promissing strategy in preparation for an influenza pandemic. DNA vaccine has been known for its ability to stimulate specific cytotoxic T cells CTL. C3D genetic adjuvants and Spdfull CD40L has been known to increase specific antibody responses. The purpose of this study is to determine the effect of the addition of genetic adjuvants C3D and Spdfull CD40L in H5N1 hemagglutinin DNA vaccines. Codon of DNA fragments of Hemaglutinin has been optimized and part transmembrane H5COP TM has been removed. Construction of H5COP TM DNA vaccine was using the expression vector pcDNA 3.1 and expressed in CHO cells was done to verify the vaccine construction. A group of 8 weeks BALB C mice were vaccinated intramuscularly with 3 weeks intervals of for each vaccination. Mice blood before primary vaccination and after each vaccination were collected and stored at 30°C to be analyzed. Another group of mice were vaccinated using plasmid pcDNA 3.1 wt as a control group, whereas four combinations of plasmid DNA, pcDNA 3.1 H5COP TM, pcDNA 3.1 H5COP TM C3D, pcDNA 3.1 Spdfull H5COP TM CD40L and pcDNA 3.1 Spdfull H5COP TM C3D CD40L were used for the treatment. Level of antibody response that occur from each treatment were measured using ELISA with H5COP as antigen coat for ELISA plate. The results of the ELISA test showed increased ratio of OD in baseline to third serum, with the highest ratio was H5COP TM C3D Spdfull CD40L 2.236, followed by H5COP TM C3D 1.900 , and the Spdfull H5COP TM CD40L 1.874.

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Vaksin DNA merupakan strategi yang menjanjikan dalam persiapan menghadapi pandemik influenza. Vaksin DNA telah diketahui kemampuannya dalam menstimulasi sel T sitotoksik yang spesifik. Adjuvan genetik C3d dan Spdfull CD40L telah diketahui dapat meningkatkan respon antibodi spesifik. Tujuan dari penelitian ini ingin mengetahui efek penambahan adjuvan genetik C3d dan Spdfull CD40L pada vaksin DNA Hemaglutinin H5N1. Fragmen DNA Hemaglutinin yang digunakan telah dioptimasi kodon dan dihilangkan bagian transmembrannya H5COP-TM. Vaksin DNA H5COP?TM dikonstruksi menggunakan vektor ekspresi pcDNA 3.1 dan hasil konstruksi setelah diverifikasi diuji ekspresi pada sel CHO. Mencit BALB/c berusia 8 minggu divaksinasi sebanyak tiga kali secara intramuskular dengan interval waktu 3 minggu untuk tiap vaksinasi. Darah mencit sebelum vaksinasi primer dan paska vaksinasi dikumpulkan dan disimpan pada -30°C untuk dianalisis. Mencit yang divaksin plasmid pcDNA 3.1 wt digunakan sebagai kelompok kontrol, sedangkan untuk perlakuan digunakan 4 kombinasi plasmid DNA yaitu, pcDNA 3.1 H5COP?TM, pcDNA 3.1 H5COP?TM-C3d, pcDNA 3.1 H5COP?TM Spdfull CD40L dan pcDNA 3.1 H5COP?TM-C3d Spdfull CD40L. Respon antibodi dari setiap perlakuan diukur menggunakan metode ELISA dengan antigen H5COP sebagai antigen pelapis pelat ELISA. Berdasarkan hasil uji ELISA, nilai rasio kenaikan OD baseline sampai serum ke-3, mencit yang divaksinasi dengan vaksin DNA H5COP?TM-C3d Spdfull CD40L memiliki nilai rasio yang paling tinggi 2.236 , diikuti dengan mencit yang divaksinasi dengan vaksin DNA H5COP?TM-C3d 1.900 , dan mencit yang divaksinasi dengan vaksin DNA H5COP?TM Spdfull CD40L 1.874."

"ABSTRAKPenelitian mengenai pengembangan vaksin DNA pengekspresi antigen fusi hemaglutinin dan VP22 terhadap respon antibodi spesifik dan sel T CD8 pada mencit BALB/c telah dilakukan. Tujuan penelitian ini adalah untuk menilai penambahan VP22 secara terfusi pada plasmid pcDNA H5cop?TM terhadap respon imun humoral dan seluler yang diinduksi oleh vaksin DNA pemgekspresi antigen hemaglutinin virus influenza A H5N1. Metodologi yang digunakan dalam penelitian ini yaitu uji eksperimental berupa kenaikan dan reaktivitas serum yang diperoleh dari kelompok mencit BALB/c yang divaksin dengan pcdnawt, pcdna-H5cop?TM, pcdna-, pcdnaH5cop?TM-VP22, pcdnaH5copfull serta respon sel T CD8 dari spleen mencit BALB/c yang mensekresikan IFN-? spesifik terhadap peptida H5N1 MHC Class I. Mencit BALB/c berusia 8 minggu divaksinasi sebanyak tiga kali secara intramuskular dengan interval waktu 2 minggu untuk tiap vaksinasi. Semua kelompok mencit menunjukkan peningkatan respon antibodi spesifik dibandingkan dengan kontrol dengan nilai rasio OD serum ketiga pada kelompok mencit pcdna-H5cop?TM, pcdnaH5cop?TM-VP22, pcdnaH5copfull dan kontrol secara berurutan adalah 1.71 p=0.006 , 1.56 p=0.010 , 1,05 p=0.016 dan 1.01. Hasil uji statistik menunjukkan bahwa tidak ada perbedaan bermakna pada kelompok perlakuan pcdna-H5cop?TM dengan pcdnaH5cop?TM-VP22 terhadap protein HA p=0.200 . Sementara pada respon sel T CD8 yang diperoleh dari optimasi ELISPOT menunjukkan adanya spot forming unit SFC pada spleen mencit yang divaksinasi dengan pcdnaH5cop?TM-VP22 pada berbagai konsentrasi peptida H5N1 yaitu berturut-turut 20 spot 100ng , 22 spot 250ng , 22 spot 500ng , 49 spot 750ng , dan 72 spot 1000ng . Nilai spot tertinggi didapatkan dengan konsentrasi peptida H5N1 sebanyak 1000ng. Hasil yang diperoleh mengindikasikan bahwa dengan adanya penambahan VP22 secara terfusi pada pcdna-H5cop?TM dapat meningkatkan respon seluler terhadap virus influenza A H5N1.

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ABSTRACTResearch on the development of DNA vaccines expressing a fused gene of haemagglutinin HA and VP22 towards specific antibody and CD8 T cells responses in mice BALB c has been done. The purpose of this study was to asses the fused VP22 into the pcDNA H5cop TM towards humoral and cellular imune responses. The methodology used in this study was experimental method that focused on increase of antibody level of serum obtained from groups of BALB c mice that previously vaccinated with pcDNAwt, pcDNA H5COP TM, pcDNA , pcDNA H5COP TM VP22, pcDNA H5COP full. Response CD8 T cell generated from spleen of mice BALB c that secreted IFN H5N1 peptides specific to MHC class I was also observed. Significant increase of level of specific antibody response were shown by value of control compared to third serum with mean value of OD optical density of pcDNA H5COP TM, pcDNA H5COP TM VP22, pcDNA H5COP full and control 1.71 p 0.006 , 1.56 p 0.010 , 1,05 p 0.015 and 1,01 respectively. Statistical analysis showed that there was no significant difference in group treated with pcDNA H5COP TM with pcDNA H5COP TM VP22 towards HA protein p 0.200 . The ELISPOT optimizations showed response to CD8 T cells by formation of spot forming units SFC in the spleen of mice vaccinated with pcDNA H5COP TM VP22 with various concentrations of peptide H5N1 applied, 20 spots 100ng , 22 spots 250ng , 22 spots 500ng , 49 spots 750ng , and 72 spots 1000ng respectively. The highest value obtained by peptide of H5N1 with a total peptide 1000ng. The results indicated that the fused of VP22 into the pcDNA H5cop TM can enhance cellular responses against H5N1 influenza A virus. "

"The present book gives an update of the “world of naked gene vaccines”, namely DNA and RNA vaccines. Its content ranges from general mechanisms, inherent immunostimulatory properties and the vast potential to modulate immune responses, to recent successful clinical studies and approved veterinary gene vaccines. Beyond the state-of-the-art of genetic immunization, the reader will be stimulated with a chapter addressing “burning questions”."

"SARS-CoV-2 sebagai virus penyebab COVID-19 yang berikatan dengan reseptor ACE-2 untuk masuk ke dalam sel inang melalui protein spike-1. Protein spike-1 dapat menjadi target pencegahan COVID-19 melalui pengembangan vaksin. Vaksin berbasis DNA merupakan kandidat vaksin yang menjanjikan untuk dikembangkan. Spesimen naso-oro faring pasien COVID-19 yang telah dikonfirmasi dengan RT-PCR, diekstraksi dan diamplifikasi dengan menggunakan primer kloning terhadap plasmid pUMVC4a. Hasil sekuensing dianalisis dengan SeqScape 3.0 dan MEGA 11. Analisis epitop sel B dilakukan dengan berbagai piranti lunak berbasis web. Konstruksi DNA vaksin dilakukan melalui analisis in silico menggunakan SnapGene 6.0 serta in vitro melalui teknik DNA Rekombinan. Gen spike-1 teramplifikasi dengan ukuran 2.265 bp, namun ligasi ke pUMVC4a dan transformasi ke E.coli strain DH5α belum berhasil. Berdasarkan analisis, seluruh sekuen memiliki mutasi D614G dengan isolat A dan B memiliki PNI yang dekat dengan varian Wuhan wt sementara 5 isolat (C-G) termasuk dalam varian Omicron. Berdasarkan sifat antigenisitas, toksisitas, alergenisitas, topologi dan hidrofobisitas, empat belas sekuen asam amino (pada posisi 68-678 protein S-1) diajukan sebagai epitop terpilih. Terdapat 14 sekuens asam amino pada protein spike-1 SARS-CoV-2 yang dapat diajukan sebagai domain epitop sel B dalam pengembangan vaksin COVID-19 berbasis DNA.

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SARS-CoV-2 as the virus that causes COVID-19 binds to the ACE-2 receptor to enter host cells via the spike-1 protein. Spike-1 protein can be a target for preventing COVID-19 through vaccine development. DNA-based vaccines are promising vaccine candidates to be developed. Naso-oropharyngeal specimens of COVID-19 patients confirmed by RT-PCR were extracted and amplified using clone primers against the plasmid pUMVC4a. The sequencing results were analyzed with SeqScape 3.0 and MEGA 11. B cell epitope analysis was performed with various web-based software. Vaccine DNA construction was carried out through in silico analysis using SnapGene 6.0 and in vitro using Recombinant DNA techniques. The spike-1 gene was amplified with a size of 2,265 bp, but ligation to pUMVC4a and transformation to E.coli strain DH5α were not successful. Based on the analysis, all sequences have the D614G mutation with isolate A and B having a PNI that is close to the Wuhan wt variant while 5 isolates (C-G) belong to the Omicron variant. Based on antigenicity, toxicity, allergenicity, topology and hydrophobicity, fourteen amino acid sequences (at positions 68 - 678 of protein S-1) were proposed as selected epitopes. There are 14 amino acid sequences in the SARS-CoV-2 spike-1 protein that can be proposed as B cell epitope domains in the development of a DNA-based COVID-19 vaccine."

"Latar Belakang : Sebuah virus influenza baru berasal dari babi, muncul di Amerika Utara pada tahun 2009, dengan cepat menyebar ke seluruh dunia dan mengakibatkan pandemi influenza A 2009. Virus ini diklasifikasikan sebagai subtipe H1N1 menurut antigenisitas dari hemaglutinin (HA) dan protein neuraminidase (NA). Pandemi influenza tahun 2009 yang disebabkan virus H1N1 telah berakhir. Namun, kemungkinan terjadinya gelombang pandemi (H1N1) 2009 kedua sulit diprediksi. Vaksin DNA merupakan pendekatan baru yang sangat menjanjikan untuk vaksinasi. Vaksin ini dapat merangsang respons imun dengan batas yang sangat luas termasuk respons antibodi, respon sel T sitotoksik dan sel T helper. Dalam penelitian ini, plasmid DNA yang mengekspresikan antigen HA dan NA dari virus H1N1 diinjeksikan pada mencit Balb/C dengan berbagai komposisi dan lokasi injeksi. Imunisasi dilakukan pada mencit Balb/C untuk mengobservasi respon antibodi yang dihasilkan. Metode : Plasmid DNA yang mengekspresikan HA dan NA dan digunakan untuk imunisasi mencit Balb/C pCDNA 3.1 diperbanyak dalam E.Coli Top 10 dan dipurifikasi atau dimurnikan dengan menggunakan Qiagen Plasmid Purification. Mencit dibagi ke dalam 5 kelompok imunisasi, yaitu : kelompok pertama diimunisasi dengan vaksin pcDNA3.1-HA/pcDNA3.1, kelompok kedua diimunisasi dengan vaksin pcDNA 3.1-HA/pcDNA 3.1-NA yang dicampurkan, kelompok ketiga diimunisasi dengan vaksin pcDNA 3.1-NA/pcDNA3.1, kelompok keempat diimunisasi dengan vaksin tunggal pcDNA 3.1-HA yang disuntikan pada paha kiri dan pcDNA 3.1-NA pada paha kanan, kelompok kelima diimunisasi dengan kontrol pCDNA3.1. Respons antibodi spesifik terhadap HA diukur dengan metode ELISA. Hasil : Kelompok mencit Balb/C yang diimunisasi dengan vaksin pcDNA 3.1 HA/pcDNA 3.1 dan pcDNA 3.1-HA/ pcDNA 3.1-NA menunjukkan kenaikan titer abtibodi yang signifikan setelah diimunisasi primer dan booster ke-3.

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Background : A new influenza virus originated in pigs, appeared in North America in 2009, quickly spread throughout the world and cause pandemic influenza A 2009. The virus is classified as H1N1 subtype according to the antigenicity of the hemagglutinin (HA) and neuraminidase protein (NA). The 2009 influenza pandemic caused by the H1N1 virus has ended. However, the possibility of a wave of H1N1 pdm 2009 is difficult to predict. DNA vaccines are a very promising new approach to vaccination. This vaccine can stimulate an immune response with a very broad limits, including antibody responses, cytotoxic T cell responses and T helper cells. In this study, Plasmid DNAs espressing HA and NA antigen of influenza A H1N1 virus were injected into Balb/C mice with various compositions and site of injection. Immunization performed on Balb / C mice was performed to observe specific antibody response towards.

Methods : Plasmid DNAs expressing hemagglutinin and neuraminidase were used for Balb/C mice immunization transformed into a plasmid pCDNA3.1. Plasmid pCDNA 3.1 that express hemagglutinin and neuraminidase propagated in E. coli Top 10 and purified using Qiagen Plasmid Purification. Mice were divided into 5 groups of immunization, namely: the first group was immunized with pcDNA 3.1-HA/pcDNA 3.1 vaccine, the second group was immunized with pcDNA 3.1-HA/pcDNA 3.1-NA vaccine, The third group was immunized by pcDNA 3.1-NA/pcDNA 3.1 vaccine, fourth group were injected by pcDNA 3.1-HA vaccine in the left thigh by and pcDNA 3.1-NA vaccine in the right thigh and the fifth group immunized with the control pCDNA3.1.vaccine. HA-specific antibody responses measured by ELISA method.

Results : The group of Balb / C mice which were immunized with pcDNA 3.1- HA/pcDNA3.1 and pcDNA 3.1-HA/pcDNA3.1-NA vaccines show an significantly increase antibody titer after primary immunization and third booster."

"Berbagai jenis vaksin telah banyak dikembangkan untuk mengatasi dan mencegah penularan penyakit infeksi. Beberapa pendekatan teknologi perancangan vaksin telah telah mengalami perkembangan yang cukup pesat untuk mengatasi masalah-masalah yang berhubungan dengan kerumitan penanggulangan penyakit infeksi. Vaksin generasi pertama yang menggunakan mikroba patogen yang dilemahkan telah banyakdigunakan, namun karena pertimbangan keamanan dari vaksin generasi pertama ini maka vaksin generasi kedua yang menggunakan mikroba patogen yang dimatikan, telah dikembangkan. Demikian pula dengan vaksin generasi ketiga yaitu vaksin rekombinan yang terdiri dari protein yang dimurnikan telah dikembangkan dan digunakan. Kemajuan dalam bidang biologi molekuler dan rekayasa genetika telah memungkinkan untuk mengembangkan vaksin generasi keempat yaitu vaksin DNA. Dalam review ini akan dibahas tentang konstruksi dan elemen genetik vaksin DNA, keuntungan dan berbagai kemajuan yang telah dicapai dalam penelitian tentang vaksin DNA.

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AbstractVaccines have been developed for a range of different infectious diseases. The complexity of microbial infections requires novel approaches to vaccine design. The first-generation of vaccines were live attenuated pathogens. Because of safety concerns, the second-generation of vaccines, chemically or physically inactivated pathogens were later developed. Purified or synthetic proteins represent a third generation, and recent advances in molecular biology and genetic engineering have led to the development ofthe fourth vaccine generation, which includes DNA and virus vector-based vaccines. This review discusses on the genetic elements and construction of DNA vaccines, comparison of DNA vaccines and conventional vaccines, the benefits and limitations of DNA vaccines, and the advances of genetic vaccine development over the lastdecade."

"ABSTRAKLatar Belakang : Penyakit demam dengue dan demam berdarah dengue yang disebabkan oleh virus dengue masih menjadi masalah kesehatan di dunia. Hingga saat ini pengobatan spesifik serta vaksin untuk infeksi dengue belum tersedia, dan berbagai strategi pembuatan vaksin sedang dikembangkan oleh berbagai pihak. Sebagai salah satu negara endemis infeksi dengue, Indonesia juga perlu melakukan pengembangan vaksin dengue dengan menggunakan strain virus yang berasal dari Indonesia. Pada penelitian ini akan dikembangkan kandidat vaksin DNA rekombinan dengan gen insersi Premembran dan Envelope virus dengue tipe 2 sebagai bagian dari pengembangan vaksin dengue tetravalen di Indonesia.Metode : Plasmid DNA rekombinan dirancang mengandung gen premembran dan envelope dari virus dengue tipe 2 isolat Indonesia. Fragmen DNA diinsersi ke dalam vektor plasmid pUMVC4a serta ditransformasi ke dalam sel E.coli DH5-α. Klon plasmid yang didapat dikonfirmasi dengan metode PCR, enzim restriksi dan sekuensing. Ekspresi protein dari plasmid diuji melalui metode transfeksi pada sel Vero. Selanjutnya plasmid disuntikkan pada mencit jenis Balb/C sebanyak 3 kali dengan interval 3 minggu pada daerah intramuskular. Penyuntikan menggunakan 2 metode : suntikan intramuskular dengan jarum (IM) dan suntikan dengan menggunakan alat needle-free injector (NFI) dengan menggunakan 2 macam dosis plasmid, yaitu 25 μg dan 100 μg DNA. Pemeriksaan antibodi dilakukan dengan metode ELISA dan PRNT. Setelah fase imunisasi selesai, dilakukan uji tantang dengan menyuntikkan 2,5 x 105 PFU/ml DENV-2 secara intraperitoneal pada beberapa kelompok mencit untuk melihat pembentukan sel B memori. Data antibodi yang diperoleh dianalisis secara deskriptif dan analitik.Hasil : Plasmid rekombinan pUMD2 telah berhasil diperoleh. Transfeksi pada sel Vero menunjukkan adanya ekspresi protein intra dan ekstraselular melalui pemeriksaan imunostaining dan ELISA. Melalui pemeriksaan ELISA, antibodi terdeteksi hanya pada kelompok penyuntikan NFI (p<0,005). Titer antibodi tertinggi dijumpai pada kelompok penyuntikan dengan NFI dosis 100 μg , kemudian NFI 25 μg dengan perbedaan yang tidak bermakna (p>0,005) antara kedua kelompok tersebut. Melalui PRNT 70% ditemukan bahwa antibodi netralisasi terhadap DENV-2 terbentuk pada mencit yang diberi imunisasi dengan metode NFI, sedangkan pada kelompok IM titernya tidak terdeteksi (<1/10). Titer antibodi terbaik diperoleh pada kelompok penyuntikan NFI dosis 100 ug yaitu 1/80-1/160. Titer hari ke-4 dan 8 setelah penyuntikan 2,5 x 105 PFU/ml virus pada kelompok yang diimunisasi secara IM dan NFI mengalami peningkatan. Sebaliknya, pada kelompok yang tidak diberi virus tidak terdapat peningkatan titer netralisasi. Hal ini menunjukkan adanya pembentukan sel B memori dari imunisasi yang diberikan.Kesimpulan : Pembuatan plasmid rekombinan dengan gen insersi pre-M dan E DENV2 strain DS18/09 telah berhasil dilakukan. Terdapat respon antibodi netralisasi dan anamnestik dari mencit yang diberi imunisasi secara NFI, namun imunisasi secara IM hanya menunjukkan respon antibodi anamnestik dari sel memori. Plasmid DNA rekombinan ini memiliki potensi sebagai kandidat vaksin DNA terhadap virus dengue tipe 2. Perlu dilakukan penelitian selanjutnya berupa rancangan vaksin tetravalen dalam upaya pengembangan vaksin dengue secara menyeluruh.

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ABSTRACTIntroduction : Dengue fever and dengue haemorrhagic fever caused by dengue virus is still a major health problem. There are four types of Dengue virus which antigenically distinguished : DEN-1, DEN-2, DEN-3, DEN-4. Currently, no specific treatment for Dengue infection and no vaccine are available, and various strategies have been used to develop dengue vaccine. Indonesia as one of dengue-endemic country has to attempt dengue vaccine development particularly using Indonesian virus strain. In this study, recombinant DNA vaccine candidate using DENV-2 pre-membrane and envelope genes was constructed as a part of dengue tetravalent vaccine development in Indonesia.Methods : The recombinant plasmid consisting pre-membrane and envelope genes from DENV-2 Indonsia isolate was constructed. DNA fragment were inserted to pUMVC4a plasmid vector and then transformed to E. Coli DH5-α. The construction was confirmed using PCR, restriction enzyme and sequencing. Protein expressions of preM and E were determined by transfection into Vero cells. Group of Balb/C mice were injected with amount of plasmid via intra muscular route. The injection was conducted using 2 delivery methods : conventional syringe-needle (IM) and needle-free injector (NFI) device. Doses of plasmid that being compared are 25 μg and 100 μg. Mice were immunized with plasmid 3 times with 3 weeks interval. Antibody titre were determined by ELISA and PRNT. After immunization phase, part group of mice challanged with 2,5 x 105 PFU/ml DENV-2 intra peritoneally to confirm wether immunization induced memory cells. Antibody data were interpretated by descriptive and analytic methodsResults : The recombinant plasmid pUMD2 has been constructed. Confirmation of gene secuence showed no mutation at the clone. Vero cells-81 transfected with pUMD2 expressed prM and E as determined by immunofluorescence staining as intracellular protein and by ELISA to detect extracellular protein. In ELISA results, antibody was detected only in NFI group (p<0,005). Highest antibody titre was found in NFI group with dose 100 μg followed by dose 25 μg. However, antibody titre by ELISA between NFI group dose 100 μg and 25 μg were not statistically significant (p>0,005). Neutralizing antibody by PRNT 70% showed concordant result compared to ELISA. Neutralizing titre to DENV-2 was developed in NFI group, but it was not detectable in IM group of mice (<1/10). The highest titre of neutralization achieved by 100 μg, NFI group whose titer 1/80-1/160. Immunized mice in all groups raised greater neutralizing antibody titers on days 4 and 8 after challange with 2,5 x 105 PFU/ml of DS 18/09 of dengue type 2 virus. Compared to immunized mice that were not challanged which developed no increasing neutralizing titre, it indicated that immunization could produced memory B cell responses.Conclusions : Recombinant plasmid as a candidate for dengue DNA vaccine has been constructed. The plasmid expressed premembrane and envelope proteins in Vero cells. Immunogenicity test from the plasmid DNA demonstrated neutralizing antibody responses and anamnestic responses in mice which immunized only by NFI method. IM injection method only showed anamnestic antibody responses. Overall, this DNA plasmid has a potency to be used as a candidate for DNA vacine against dengue virus type-2. Study for designing tetravalent vaccine model is necessary as a part in vaccine dengue development."

"Infeksi human papillomavirus tipe 16 (HPV16) dapat menyebabkan kanker servikk, penyebab kematian no 2 di dunia. Salah satu pencegahannya adalah dengan imunisasi. Vaksin komersil saat ini merupakan vaksin viral like particles (VLP) diproduksi pada sistem ekspresi yeast dan baculovirus. Sebagai upaya penyediaan vaksin dengan harga lebih ekonomis telah dilakukan pengembangan vaksin DNA dan protein rekombinan L1 HPV16 yang diekspresikan di prokariota. Antigenitas vaksin DNA dan L1 rekombinan diujikan dalam peneltian ini. Penelitian dimulai dari pemindahan L1 dari pUC L1 HPV16 ke pCDNA3.1, ekspresi L1 rekombinan dalam prokariota, pengamatan pembentukan VLP oleh L1 rekombinan dengan transmission electron microscope (TEM), pengujian antigenitas kombinasi vaksin DNA dan protein rekombinan pada BALB/c. Hasil menunjukkan pcDNA3.1 L1 berhasil diperoleh yang dibuktikan dengan analisis enzim restriksi dan sekuensing. L1 rekombinan berhasil membentuk VLP, vaksin komposisi 12,5 μg pcDNA3.1 L1 dikombinasikan 2 μg L1 rekombinan menginduksi titer antibodi endpoint tertinggi pada pengambilan serum terakhir yaitu 23.55 (p<0.05) dibandingkan vaksin 12,5 μg pcDNA3.1 L1 (2.775) dan 2 μg L1 rekombinan (10.45) yang diberikan secara terpisah setelah 3 kali imunisasi. Sebagai kesimpulan pCDNA3.1L1 berhasil diperoleh, protein L1 rekombinan dapat membentuk VLP dan pemberian kombinasi pcDNA3.1 L1 dan L1 rekombinan menginduksi respon kekebalan tubuh lebih bagus dibandingkan pemberian secara terpisah.

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Infection with human papillomavirus type 16 (HPV16) can cause cervical cancer, the second cause of death in the world. One way to prevent it is with a barrier. The current commercial vaccine is a viral like particle (VLP) vaccine produced on yeast and baculovirus expression systems. As an effort to provide a vaccine at a more economical price, a DNA vaccine and a recombinant L1 HPV16 protein that are expressed in prokaryotes have been developed. The antigenicity of recombinant DNA and L1 vaccines was tested in this study. The research started with the transfer of L1 from pUC L1 HPV16 to pCDNA3.1, expression of recombinant L1 in prokaryotes, assessment of VLP formation by recombinant L1 with transmission electron microscopy (TEM), testing the antigenicity of a combination of DNA vaccines and recombinant protein on BALB/c. The results showed that pcDNA3.1 L1 was successfully obtained, as evidenced by restriction enzyme analysis and sequencing. The recombinant L1 succeeded in forming a VLP, the composition of the vaccine 12.5 µg PCDNA3.1 L1 combined 2 µg L1 recombinant induced the highest endpoint antibody titer (10.45) which was given 23.55 (p <0.05) compared to the 12.5 µg vaccine PCDNA 3.1 L1 (2,775) and 2 µg L1 recombinant (10.45) given by 3 times. As a conclusion, pCDNA3.1L1 was successfully obtained, recombinant L1 protein can form VLP and provide a combination of recombinant pcDNA3.1 L1 and L1 induce an immune response better than administration separately."