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BACKGROUND: BCG vaccine has been proven to be effective protection against tuberculosis (TB) meningitis and miliary TB. However, for protection against pulmonary TB, the results remains vary widely. Recombinant vaccine consisting of two immunodominant Mtb antigens ESAT-6-Ag85C-polyHistag (EAH) is currently being developed as a new TB vaccine candidate for booster. An immugonecity test for vaccine candidates is required in the initial phase to evaluate cellular immune response. This study was conducted to evaluate the cellular immune response by measuring interferon-gamma (IFN-γ) cytokines produced by T cells after ex vivo stimulation by TB EAH antigen fusion.METHODS: Peripheral blood mononuclear cells supernatant samples were collected from 16 new pulmonary TB subjects, 17 pulmonary TB in treatment subjects, and 10 healthy subjects. Samples were tested for IFN-γ level with enzyme-linked immunosorbent assay (ELISA). Kruskal-Wallis test was used to test the differences between IFN-γ levels among three groups, and followed by post-hoc analysis using Mann Whitney.RESULTS: The median of IFN-γ levels for new pulmonary TB, pulmonary TB in treatment, and healthy subjects were 17.09 (2.65-140.14) pg/mL, 4.36 (2.43-21.41) pg/mL, and 2.91 (2.39-3.85) pg/mL, respectively. There were significant differences of IFN-γ levels between new pulmonary TB group and pulmonary TB in treatment group (p=0.012), between new pulmonary TB group and healthy group (p=0.001), and also between pulmonary TB in treatment group and healthy group (p=0.035).CONCLUSION: Results show that TB EAH could stimulate cell-mediated immune responses in the three groups, with the highest IFN-γ levels are found in new pulmonary TB group, suggesting a potential immunodominant antigen fusion for vaccine candidate development.KEYWORDS: Ag85C, ESAT-6, immunogenicity test, IFN-γ, TB vaccine candidate, tuberculosis

Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

Scaling up SARS-CoV-2 testing and tracing continues to be plagued with limitation of sample collection method that requires trained healthcare workers to perform and cause discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days on room temperature storage. The developed method was validated for use on multiple RNA extraction kit and commercially available COVID-19 RT-PCR kits. Our developed method achieved sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35) with 95.16% of patients prefer the self-collected gargle method.

Background We explored the outcome of convalescent plasma (CP) treatment in patients with moderate and severe coronavirus disease 2019 (COVID-19) and investigated variables for the design of further trials in Indonesia. Methods Hospitalised patients with moderate (n = 5) and severe (n = 5) COVID-19 were recruited and transfused with CP from donors who recovered from mild (n = 5), moderate (n = 5), or severe (n = 1) COVID-19. Neutralising antibodies (NAbs) to the virus were measured at the end of the study using a surrogate virus neutralisation test as an alternative to the plaque reduction assay. Clinical improvement was assessed based on the modified World Health Organization Research and Development Blueprint six-point scale, Brixia Chest-X-Ray scoring, and laboratory parameters. The study was registered at ClinicalTrials.gov (NCT04407208). Findings CP transfusion in three doses of 3 mL/kg of recipient body weight at 2-day intervals was well tolerated. Good clinical improvement was achieved in all patients with moderate disease and in two patients with severe disease. Most patients at baseline had detectable NAbs with median inhibition rates comparable to those of the donors (90·91% vs. 86·31%; p = 0·379). This could be due to the unavailability of pre-donation NAb testing and postponed CP administration that required communal consent. Interpretation This study highlights the safety of CP therapy. Although improvements were observed, we could not conclude that the outcomes were solely due to CP treatment. Further randomised controlled trials that cover different disease stages with pre-donation NAb measurements using locally applicable strategies are warranted. Funding The study was supported by PT Bio Farma, Indonesia.

Recombinant small hepatitis B surface antigen (sHBsAg) is used as a vaccine component to prevent hepatitis B virus infection. As an attempt to produce local recombinant sHBsAg, a PCR-amplified DNA fragment encoding Indonesia sHBsAg which belongs to B genotype and adw2 subtype was cloned into Hansenula polymorpha expression vector pHIPX4 by using recombination method. The resulted pHIPX4-sHBsAg was integrated into the alcohol oxidase (AOX) locus of H. polymorpha NCYC495 genome and the sHBsAg expression was regulated under the control of H. polymorpha AOX promoter. H. polymorpha NCYC495 carrying the sHBsAg coding sequence was grown in mineral medium and methanol 0.5% (v/v) was added to induce the expression of recombinant sHBsAg. The expression of sHBsAg was detected by HBsAg diagnostic kit test, ELISA, and Western blot analysis.

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.

Antiviral nucleos(t)ide analogue (NUCs) merupakan pengobatan utama pada hepatitis B kronis (HBK). Pemberian jangka panjang dinilai cukup efektif menekan progresivitas penyakit, namun dapat menimbulkan mutasi resisten. Studi ini melihat karakteristik gen polimerase yang berkaitan dengan resistensi NUCs pada penderita HBK asimptomatik pra-pengobatan. Penelitian dilakukan di Laboratorium Hepatitis, Lembaga Biologi Molekuler Eijkman, Jakarta. Sebanyak 38 sampel individu dengan hepatitis B surface antigen (HBsAg) positif dikarakterisasi dengan PCR-sekuensing. Genotipe dan subtipe ditentukan berdasarkan sekuens HBsAg. Sebanyak 37 (97,4%) sampel menunjukkan mutasi rtQ238H/N dan satu sampel wildtype. Sebanyak 23 (62,2%) memiliki mutasi rtQ238H, 10 (27,0%) rtQ238N, dan empat (10,8%) dengan mutasi ganda rtA194T dan rtQ238H. Genotipe B ditemukan pada 26 (68,4%) sampel, genotipe C pada 11 (28,9%), dan genotipe D pada satu (2,6%) sampel. Secara statistik, mutasi rtQ238H berasosiasi dengan genotipe B (p

The hepatitis B virus (HBV) genotype is associated with viral anthropological history, clinical outcome of disease and response to treatment. This study examines the HBV genotypes in Indonesia. HBV genotypes were determined by whole-genome sequencing and from the sequence of the Pre-S2 and S regions in a larger series. Two HBV genotypes, B (HBV/B) and C (HBV/C), were predominant. Three previously reported HBV/B subgenotypes were identified, with certain population association: HBV/B2 (HBV/Ba) was found mostly in Indonesians of Chinese ethnic origin, HBV/B3 was dominant among the Javanese, and HBV/B5, reported earlier from the Philippines, was also discovered, albeit at low frequency. Two other subgenotypes, HBV/B4 from Vietnam and HBV/B6, recently reported from the Arctic region, were not found. A novel subgenotype, HBV/B7, was recognized, associated with populations of the Nusa Tenggara islands in eastern Indonesia. Characteristic differences in HBsAg serotype and single nucleotide polymorphisms (SNPs) in the Pre-S2 region distinguish HBV/B7 from other HBV/B subgenotypes and further establish the new HBV subgenotype.