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"Infeksi HIV-1 merupakan salah satu permasalahan kesehatan di Indonesia yang perlu diteliti untuk mendapatkan strategi intervensi yang sesuai dengan galur virus yang bersirkulasi di Indonesia. Untuk pengembangan kultur galur HIV Indonesia, diperlukan antibodi spesifik terhadap protein awal HIV-1 yaitu Tat dan Rev sebagai marka infeksi HIV pada sel yang terinfeksi HIV-1. Penelitian ini bertujuan untuk mendapatkan plasmid pengekspresi bagian immunodominant protein fusi Tat dan Rev pada sistem ekspresi mamalia agar dapat dimanfaatkan untuk menginduksi pembentukan antibodi spesifik pada hewan coba mamalia. Susunan DNA penyandi protein fusi Tat dan Rev didapat dengan melakukan analisis optimasi kodon penyandi susunan asam amino protein fusi menggunakan piranti lunak GenScript, untuk mendapatkan nilai Codon Adaptation Index CAI > 0.80 pada sistem ekspresi mamalia. Susunan nukleotida yang diperoleh kemudian diberikan tambahan susunan nukleotida situs restriksi pada bagian 5 rsquo; dan 3 rsquo; untuk memudahkan pengklonaan ke dalam vektor ekspresi dan dipesan ke penyedia jasa sintesis asam nukleat. Potongan DNA sisipan TatRev, penyandi protein fusi TatdanRev diperoleh dari penyedia jasa sintesis dalam bentuk terklona dalam plasmid pUC19. Agar dapat terekspresi dalam sistem ekspresi mamalia, DNA sisipan dipotong dengan enzim restriksi KpnI dan HindIII dari plasmid pUC19 dan disubklona ke dalam situs restriksi KpnI dan HindIII pada plasmid pTriEx-4. Analisis restriksi enzim Kpn I dan Hind III plasmid rekombinan yang diperoleh dengan elektroforesis jel agarosa menunjukkan adanya dua pita yang bermigrasi sesuai ukuran plasmid pTriEx-4 dan DNA sisipan TatRev, yaitu sebesar 5000 pb dan 600 pb.

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HIV 1 infection is one of the health problems in Indonesia that is necessary to be studied in order to obtain intervension strategies that is in accordance with the circulating viral strains in Indonesia. In order to develop culture of Indonesian HIV strainss, specific antibodies towards early proteins of HIV, namely Tat and Rev, that are markers of HIV infection in HIV 1 infected cells. This study is aimed at obtaining a plasmid for expression of immunodominant region of Tat and Rev fusion protein in mammalian expression system to be used for stimulation of specific antibody formation in mammals as experimental animal. Nucleotide sequence of DNA encoding the Tat and Rev fusion protein was obtained by codon optimization analysis of the amino acid sequence of the fusion protein using the GenScript software, to obtain a Codon Adaptation Index CAI 0.80 for mammalian expression system. The nucleotide sequence that is obtained was then added with recognition sequences for enzymatic restriction at the 5 rsquo and 3 rsquo end to facilitate cloning into expression vector, and requested to a service provider for nucleic acid synthesis. The TatRev insert DNA, encoding the fusion protein of Tat and Rev was obtained from the nucleic acid synthesis service provider in the formed of cloned DNA in the plasmid pUC19. For expression in mammalian expression system, the insert DNA was restricted using restriction enzymes KpnI and HindIII from the pUC19 plasmid and subcloned into the KpnI and HindIII restriction sites in plasmid pTriEx 4. Agarose gel electrophoresis analysis of KpnI and HindIII enzymatic restriction of the resulting recombinant plasmid showed the presence of two bands that migrated according to the sizes of the plasmid pTriEx 4 and the TatRev insert DNA, namely 5000 bp and 600 bp."

"Men who have sex with men living with HIV (MSM-LWH) experience psychological and social issues, including depression, anxiety, fear of infecting others, frustration, and social isolation. They may also experience problems in their relationships due to a fear of social stigma, such as marital issues, family conflicts, a lack of family support, economic difficulties, and social rejection by the family. This research aimed to assess the relationship between HIV status disclosure and stress in MSM-LWH in Medan, Indonesia. Here, a cross-sectional design and the convenience sampling technique were used. A total of 176 respondents who were MSM, HIV positive, and residents of Medan City were included in this work. Data were collected by means of HIV Status Disclosure questionnaires and a Perceived Stress Scale (PSS). Overall, 70.9% respondents reported disclosing their status to others and approximately half revealed experiencing stress. Moreover, HIV status disclosure was significantly associated with stress (p= 0.025). This study reveals that HIV status disclosure may result in negative effects on MSMLWH, represent a barrier to medical treatment, and increase internal stress."

"Latar Belakang: P24 protein adalah salah satu protein milik HIV-1 yang membentuk kapsid bagian dalam dan dirilis ketike Gag protein di belah oleh protease virus. Kadar antien p24 umumnya akan meningkat di darah pada fase akut infeksi. Oleh sebab ini, produksi protein rekombinan p24 yang efektif dan memiliki imunogenisitas serta imunoreaktifitas seperti protein p24 natural merupakan hal yang penting, untuk deteksi antibodi p24 dan membuat antibodi monoklonal p24. Dalam penelitian ini, dilakukan optimasi ekspresi protein p24 subtype HIV-1 CRF01_AE. Metode : Penelitian ini menguji beberapa factor untuk ekspresi optimal dari protein rekombinan p24 pada plasmid pQE-80L milik Eschericia coli, termasuk konsentrasi inductor, durasi induksi, dan kultur media yang digunakan. Deteksi, quantifikasi, dan analisis protein dilakukan dengan SDS Page dan di analisis menggunakan Imagelab. Hasil : p24 recombinant protein from HIV-1 Subtype CFR01_AE terekspresikan secara optimal menggunakan media Terrific Broth dengan di induksi 1mM Isopropyl-beta-D-thiogalactopyranoside (IPTG) selama 6 hours pada suhu 37°lC. Kesimpulan : Strategi dan Metode yang digunakan untuk mengekspresikan protein rekombinan dapat berbeda antar sistem dan antar jenis protein yang diekspresikan. Penelitian ini menunjukkan bahwa plasmid pQE80L-p24 yang sudah dikembangkan di PRVKP FKUI memiliki kemampuan untuk mengekspresikan protein rekombinan p24, dan teroptimasi pada media ekspresi Terrific Broth, konsentrasi Isopropyl-beta-D- thiogalactopyranoside (IPTG) 1mM, selama 6 jam pada suhu 37°C.

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Background: P24 protein is one of HIV-1 protein that forms inner capsid and is released during cleave of Gag protein by viral protease. P24 antigen level will increase in the blood during the acute stage of infection. Therefore, an effective production of p24 recombinant protein that have the same immunogenicity and immunoreactivity of natural p24 protein are very important for detecting of p24 antibody and producing monoclonal p24 antibody. In this research, expression of p24 obtained from HIV-1 Subtype (HIV-1 CRF01_AE) was optimized to find the best expression system for the above protein.

Methods: In this research, several factors will be tested for optimal expression of recombinant p24 protein in plasmid pQE-80L of Eschericia coli, including the inducer concentration, duration of induction, and culture medium. Detection, quantitation, and analysis of the protein will be done using SDS Page and documented using Gel Documentation System to compare the effect of these factors.

Result: p24 recombinant protein from HIV-1 Subtype CFR01_AE is optimally expressed in Terrific Broth Medium in 1mM Isopropyl-beta-D-thiogalactopyranoside (IPTG), for 6 hours.

Conclusion: The strategies and method for protein expression may vary between a system, and a protein, to another. From this research it could be concluded that pQE80L-p24 plasmid that has been developed in PRVKP FKUI is able to express p24 recombinant protein. Protein expression is optimized in Terrific Broth Medium in 0.5mM Isopropyl-beta-D-thiogalactopyranoside (IPTG), for 4 hours in 37°C."

"Protein pol termasuk dalam tiga gen signifikan yang mengkode protein struktural HIV-1. Namun, sebagian besar tes diagnostik HIV hanya melibatkan dua produk HIV yang signifikan yaitu, protein Env atau Gag, yang berarti bahwa sebagian besar tes diagnosa HIV dilakukan atau dikembangkan menggunakan antigen yang sama. Hal ini pada akhirnya dapat menghasilkan hasil positif palsu yang berulang jika ada antibodi yang bereaksi silang karena sebagian besar tes disiapkan hanya dengan antigen umum yang sama. Solusi untuk masalah ini adalah mengembangkan uji diagnostik alternatif yang berbeda menggunakan antigen utama lain, seperti protein Pol. Salah satu produk protein Pol, enzim Integrase, termasuk dalam salah satu protein imunogenik HIV, yang berarti dapat digunakan untuk meningkatkan spesifisitas tes diagnostik HIV. Menggunakan protein Pol Immunodominant 2 (Pol ID2), sebuah protein yang terdiri dari Integrase dan RNAse H, yang sudah dikembangkan sebelumnya menggunakan subtipe HIV-1 yang paling menonjol di Indonesia (HIV-1 CRF01_AR), penelitian ini bertujuan untuk mendapatkan pengetahuan tentang kondisi optimal untuk mengekspresikan protein rekombinan Pol ID2 dalam plasmid pQE-80L Escherichia coli (E. coli) dengan harapan dapat berkontribusi terhadap pengembangan dan peningkatan kemandirian tes diagnostik HIV-1 di Indonesia. Metode: Penelitian ini dilakukan menggunakan metode desain studi eksperimental analitik. Dalam penelitian ini, protein rekombinan Pol Immunodominant 2 (ID2) dalam plasmid pQE-80L E. coli diekspresikan pada beberapa variabel media kultur, konsentrasi penginduksi, dan waktu induksi. Kultur ekspresi divisualisasikan menggunakan elektroforesis SDS PAGE dan didokumentasikan menggunakan mesin ImageQuant Las 4000. Meskipun tidak ada analisis statistik yang dilakukan, software analisis gel Image Lab 6.1 digunakan untuk menganalisis, mengukur, dan mendapatkan rasio kuantitas absolut dari konsentrasi protein pada setiap variabel. Hasil: Protein rekombinan Pol ID2 yang diperoleh dari HIV-1 subtipe CFR01_AE dapat diekspresikan secara optimal menggunakan media Terrific Broth dengan menggunakan 1mM Isopropil- beta-D-thiogalactopyranoside (IPTG) selama 3 jam induksi. Kesimpulan: Dapat disimpulkan bahwa plasmid pQE80L-Pol ID2 yang sebelumnya sudah dikembangkan di laboratorium PRVKP FKUI-RSCM dapat mengekspresikan protein rekombinan Pol ID2 dari HIV-1 subtipe CFR01_AR yang dikembangkan di bawah laboratorium yang sama. Protein dapat diekspresikan secara optimal dalam media kultur Terrific Broth menggunakan IPTG 1mM selama 3 jam induksi pada suhu 37oC.

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Background: Pol protein is included in the three significant genes that encode a structural protein of HIV-1. However, most HIV diagnostic tests involve only the two significant HIV products, Env or Gag protein, which means that most manufacturers use the same antigen sequence to develop these tests. This may eventually result in repetitive false-positive results if there is any cross-reacting antibody since the test is prepared only with the same common antigens. A solution to this problem is developing a different alternative diagnostic assay using a different major antigen such as Pol genes. One of the Pol gene products, viral enzyme integrase, is included in one of the immunogenic proteins of HIV, meaning that it may be used to improve the specificity of HIV diagnostic assays. Using a previously generated Pol Immunodominant 2 (Pol ID2) protein, comprised of Integrase and RNAseH, obtained from the most prominent HIV-1 subtype in Indonesia (HIV-1 CRF01_AR), this research aims to gain knowledge regarding the optimal conditions to express Pol ID2 recombinant protein in pQE-80L plasmid of Escherichia coli (E. coli) in hopes to contribute towards the development and self-reliance of HIV-1 diagnostic tests in Indonesia. Experimental, analytical study design was used for this research. The Recombinant Pol Immunodominant 2 (ID2) protein in the pQE-80L plasmid of E. coli was expressed under several variables of culture media, inducer concentration, and induction time. The expression culture was visualized using SDS PAGE and documented using ImageQuant Las 4000 machine. Although no statistical analysis was done, Image Lab 6.1 gel analysis software was used to analyze, quantify, and obtain the absolute quantity ratio of protein concentration of each variable.

Result: Pol ID2 recombinant protein obtained from HIV-1 subtype CFR01_AE are optimally expressed using Terrific Broth media using 1mM Isopropyl-beta-D-hiogalactopyranoside (IPTG) for 3 hours of induction.

Conclusion: It could be concluded that pQE80L-Pol ID2 plasmid previously developed in IHVCB FMUI-RSCM Laboratory can express Pol ID2 recombinant protein from HIV-1 subtype CFR01_AR that is constructed under the same laboratory. The protein expression is optimized in Terrific Broth culture media using 1mM IPTG inducer for 3 hours of induction in 37oC."

"ABSTRACTBackground: the global scale-p of antiretroviral therapy (ART) is the primary factor contributing to the decline in deaths from acquired immune deficiency syndrome (AIDS)-related illnesses. However, the emergence of transmitted drug resistance (TDR) compromises the effects of ART in treatment-naive individuals, which may hinder treatment success. The present study aimed to identify the presence of TDR among treatment-naive individuals in Buleleng, Bali, which is currently ranked sixth among Indonesian provinces with the highest cumulative human immunodeficiency virus type 1 (HIV-1) infection cases. Methods: thirty-nine ART-naive individuals in Buleleng Regency General Hospital were enrolled in the present study. Blood samples from participants were subjected to a genotypic analysis. Results: 28 protease (PR) and 30 reverse transcriptase (RT) genes were successfully amplified and sequenced from 37 samples. HIV-1 subtyping revealed CRF01_AE as the dominant circulating recombinant form in the region. No TDR for PR inhibitors was detected; however, TDR for RT inhibitors was identified in five out of 30 samples (16.7%). Conclusion: these results indicate the emergence of TDR among ART-naive individuals in Buleleng, Bali. This issue warrants serious consideration because TDR may hamper treatment success and reduce ART efficacy among newly diagnosed individuals. Continuous surveillance with a larger sample size is necessary to monitor TDR among ART-naive individuals."

"Latar Belakang: gp41 adalah transmembran glikoprotein yang memiliki peran penting dalam fusi dan masuknya Human Immunodeficiency Virus-1 (HIV-1). Keterlibatan protein transmembrane gp41 sangatlah krusial di seluruh fase awal penularan mukosa HIV-1; maka keberadaan protein ini penting untuk skrining dan diagnosis HIV-1. Protein transmembran gp41 dapat dimanfaatkan sebagai alat diagnostik HIV-1 melalui uji deteksi antibodi yang meliputi, Rapid Diagnostic Test, ELISA, dan Western Blot (WB). Namun, meskipun terdaftar sebagai salah satu protein HIV-1 yang memiliki banyak manfaat, studi mengenai ekspresi dan optimalisasi protein transmembran gp41 masih kurang diteliti. Oleh karena itu, penelitian ini bertujuan untuk memperoleh pengetahuan mengenai kondisi optimal untuk mengekspresikan protein transmembran gp41 pada Escherichia coli (E. coli) PQE80L untuk pengembangan uji diagnostik HIV-1. Metode: Penelitian ini menggunakan bagian imunodominan dari protein transmembrane gp41 (gp41-IDR). Protein gp41-IDR kemudian diekspresikan dalam sistem ekspresi E. coli dan dioptimalkan pada berbagai variabel, yaitu media kultur, konsentrasi penginduksi dan waktu induksi. Hasil yang diperoleh dari SDS-PAGE 20% didokumentasikan oleh ImageQuant Las 4000, sedangkan kuantisasi dan analisa protein gp41-IDR dilakukan di Image Lab 6.1. Software for Mac. Hasil: Hasil penelitian menunjukkan bahwa protein gp41-IDR lebih maksimal jika diekspresikan pada medium Terrific Broth (TB) dibandingkan dengan dua media kaya nutrisi lainnya, yaitu Luria Bertani (LB) dan 2x Yeast Extract-Tryptone (2x YT). Di antara lima konsentrasi Isopropil-beta-D-thiogalactopyranoside (IPTG) yang diuji, induksi oleh 1 mM IPTG menunjukkan hasil protein tertinggi jika dibandingkan dengan konsentrasi IPTG lainnya. Apabila dibandingkan dengan protein yang diinduksi selama 6 jam dan semalam, induksi protein gp41-IDR rekombinan selama 3 jam menunjukkan hasil protein tertinggi. Kesimpulan: Protein rekombinan gp41-IDR HIV-1 berhasil diekspresikan pada Escherichia coli (E. coli), dengan PQE80L sebagai vektor ekspresi. Hasil dari penelitian ini menunjukkan bahwa protein rekombinan gp41-IDR dari HIV-1 Subtipe CFR01_AE diekspresikan secara optimal pada medium Terrific Broth (TB), dengan 1 mM IPTG selama 3 jam.

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Background: gp41 is a viral transmembrane glycoprotein that plays a significant role in the fusion and entry of Human Immunodeficiency Virus-1 (HIV-1). The involvement of gp41 transmembrane protein is pivotal throughout the early phases of HIV-1 mucosal transmission; hence the presence of this protein is important for HIV-1 screening and diagnosis. gp41 transmembrane protein can be utilized as an HIV-1 diagnostic tool through antibody detection tests, namely Rapid Diagnostic Test, ELISA, and Western Blot (WB). However, despite being listed as one of the most valuable HIV-1 proteins, research regarding the expression and optimization of gp41 transmembrane protein is likely underreported. Therefore, this research aims to obtain knowledge regarding the optimal condition to express gp41 transmembrane protein in Escherichia coli (E. coli) PQE80L for the development of HIV-1 diagnostic test.

Methods: The immunodominant region of gp41 transmembrane protein (gp41-IDR) was used in this study. gp41-IDR protein was expressed in E. coli expression system and optimized under varying variables, namely culture medium, inducer concentration and induction time. The results obtained from SDS-PAGE 20% were documented by ImageQuant Las 4000, while quantitation and analysis of the gp41-IDR protein was done in Image Lab 6.1. Software for Mac.

Results: The results indicated that the gp41-IDR protein yield was maximized when expressed in Terrific Broth (TB) Medium as compared to other two nutrient-rich media, namely Luria Bertani (LB) and 2x Yeast Extract-Tryptone(2x YT). Among the five Isopropyl-beta-D-thiogalactopyranoside (IPTG) concentrations tested, induction by 1 mM of IPTG showed the highest protein yield when compared to the other IPTG concentrations. In comparison to those induced for 6 hours and overnight, induction of recombinant gp41-IDR protein for 3 hours offered the highest protein yields.

Conclusion: To conclude, recombinant gp41-IDR HIV-1 protein was successfully expressed in the Escherichia coli (E. coli) host system, with PQE80L as expression vector. Findings from this study indicate that gp41-IDR recombinant protein from HIV-1 Subtype CFR01_AE is optimally expressed in Terrific Broth (TB) Medium, with 1 mM of IPTG for 3 hours."