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Abstrak :
Latar belakang: Infeksi virus dengue masih menjadi masalah di negara tropis seperti Indonesia. Infeksi virus dengue menyebabkan mortalitas dan morbiditas yang tinggi. Belum ada obat ataupun vaksin yang telah disetujui penggunaannya dan tersedia di dunia untuk penyakit infeksi dengue. Pencegahan infeksi dengue masih terbatas pada pengendalian vektor nyamuk Aedes aegypti. Keempat serotipe virus dengue beredar di Indonesia. Dua genotipe dari virus dengue tipe 1 (DENV-1) yaitu genotipe I dan IV lebih dominan bersirkulasi di Indonesia. Pada tahun 2012, kami mengembangkan kandidat vaksin DNA DENV-1 pUMVC RDS 59/09. Tujuan penelitian ini untuk mengetahui kadar antibodi anti-membran dan envelop DENV-1 yang dihasilkan mencit ddY setelah imunisasi sebanyak tiga kali dengan pUMVC RDS 59/09 dan untuk mengetahui kemampuan antibodi tersebut dalam menetralkan beberapa genotipe DENV-1 yang diisolasi di Indonesia. Metode: Penelitian eksperimental ini dimulai dari perbanyakan pUMVC RDS 59/09 di sel E.coli untuk mendapatkan konsentrasi yang tinggi. Imunisasi dilakukan pada 12 mencit strain ddY dengan pUMVC RDS 59/09 25 μg/100 μl menggunakan needle-free injector, sebanyak tiga kali dengan interval waktu 3 minggu. Sebanyak 12 mencit disediakan sebagai kontrol yang tidak diimunisasi. Antibodi anti-membran dan envelop DENV-1 pada masing-masing serum mencit diperiksa dengan ELISA dan dibaca pada panjang gelombang 450 nm. Berikutnya, pooled serum mencit pasca imunisasi ke 3, digunakan untuk netralisasi 13 isolat DENV-1 dengan metode focus reduction neutralization test (FRNT). Fokus yang didapatkan dari FRNT diwarnai dengan tehnik imunoperoksidase dan dihitung secara manual. Hasil: Rerata nilai OD ELISA antibodi anti-membran dan envelop DENV-1 dari serum mencit kelompok imunisasi yang diambil sebelum imunisasi, pasca imunisasi 1, pasca imunisasi 2 dan pasca imunisasi 3 adalah 0,329;0,843;1,524 dan 1,598, secara berurutan. Terdapat peningkatan nilai OD ELISA antibodi anti-membran dan envelop DENV-1 dari pooled serum mencit kelompok imunisasi pasca imunisasi pertama, kedua dan ketiga dibandingkan dengan baseline. Titer FRNT antibodi anti-membran dan envelop DENV-1 dari pooled serum mencit pasca imunisasi 3 dan pooled serum mencit kontrol terhadap strain DV-1 RDS 59/09 adalah 1/320 dan < 1/10. Titer FRNT 13 isolat DENV-1 oleh antibodi anti-membran dan envelop DENV-1 dari pooled serum mencit pasca imunisasi 3 berkisar dari 1/320 sampai lebih dari 1/1280. Kesimpulan: Variasi genotipe DENV-1 tidak menyebabkan perbedaan titer antibodi netralisasi yang bermakna (p = 0,222), sehingga dapat diuraikan bahwa antibodi anti-membran dan envelop DENV-1 dapat menetralkan 13 isolat DENV-1 Indonesia yang diuji. ...... Introduction: Dengue virus infection is still a burden in tropical country such as Indonesia. Dengue virus infection causes high mortality and morbidity. No drugs or vaccines are approved and available in the world for this disease. Dengue prevention is still limited to vector control. Four dengue serotypes are circulated in Indonesia. Two genotypes of Dengue virus type 1 (DENV-1), namely genotypes I and IV are found predominantly in Indonesia. Previously in 2012, we constructed pUMVC RDS 59/09, the DENV-1 DNA vaccine candidate. The objective of this study is to assess antibody level produced in ddY strain mice after three times immunization with pUMVC RDS 59/09 and to assess the antibody ability to neutralize genotypes of DENV-1 isolated in Indonesia. Methods: This experimental study was started with propagation of pUMVC RDS 59/09 in E. coli cells to produce high concentration of the DNA. Immunization was carried out with 25 μg/ 100 μl pUMVC RDS 59/09 by needle-free injector, three times in 3 weeks interval. Twelve mice were provided for control without immunization. Anti-DENV-1 membrane and envelope antibody of individual sera were examined by ELISA and absorbance value was measured by ELISA reader in 450 nm wave length. Further, pooled sera of 3rd immunization were used to neutralize 13 DENV-1 isolates by focus reduction neutralization test (FRNT) method. The focus obtained in FRNT was stained by immune-peroxides technique and counted manually. Results: ELISA OD value mean of anti-DENV-1 membrane and envelope antibody in individual ddY mice sera of immunized group before immunization, post first immunization, after second immunization and post third immunization were 0.329; 0.843; 1.524 and 1.598, respectively. An increase in ELISA OD value of anti-DENV-1 membrane and envelope antibody in ddY mice pooled sera of immunized group after first, second and third immunization compared to baseline was observed. FRNT titre of anti-DENV-1 membrane and envelope antibody from third immunization pooled sera compared to control mice pooled sera in RDS 59/09 isolate neutralization was 1/320 compared to < 1/10. Neutralization titre of 13 DENV-1 isolates by anti-DENV-1 membrane and envelope antibody from third immunization pooled sera ranged from 1/320 to more than 1/1280. Conclussions: DENV-1 genotype variation did not lead to significant neutralization antibody titre difference (p = 0,222), so it can be explained that anti-DENV-1 membrane and envelope antibody was able to neutralize 13 strains of Indonesia DENV-1 isolates examined.

Abstrak :
COVID-19 merupakan penyakit penyebab pandemi pada akhir 2019. Perbedaan manifestasi klinis pada infeksi SARS-CoV-2 ini memicu banyak pertanyaan di kalangan peneliti dan medis. Perbedaan klinis COVID-19 tersebut dapat dipicu oleh faktor hospes, patogen maupun lingkungan. Infeksi SARS-CoV-2 terutama melalui saluran napas atas, tempat kolonisasi mikroba komensal dan patogen. Bagaimana interaksi antara mikroba yang berkolonisasi dengan SARS-CoV-2 dalam menimbulkan respons inflamasi di saluran napas atas masih belum diketahui dengan jelas. Penelitian ini bertujuan menganalisis hubungan antara karakteristik mikrobiota, serta rasio kadar sitokin pro- dan anti-inflamasi dari saluran napas atas dengan beratnya COVID-19. Penelitian ini merupakan studi potong lintang menggunakan 74 swab nasofaring dan orofaring di dalam viral transport medium (VTM) dari pasien COVID-19 berusia 18–64 tahun. Profil mikrobiota di saluran napas atas dan kadar IL-6, IL-1β, IFN-γ, TNF-α dan IL-10 diperiksa dengan metode sekuensing 16S ribosomal RNA dan Luminex assay, secara berurutan. Selanjutnya dilakukan analisis hubungan antara beratnya COVID-19 dengan OTU, keragaman alfa dan beta dari mikrobiota saluran napas atas. Lima filum terbanyak di saluran napas pasien COVID-19 di Indonesia berusia 18-64 tahun adalah Firmicutes (32,3%), Bacteroidota (27,1%), Fusobacteriota (15,2%), Proteobacteria (15,1%) dan Actinobacteria (7,1%). Analisis indeks Shannon dan ACE menunjukkan bahwa tidak ada penurunan keragaman microbiota saluran napas atas dengan bertambah beratnya penyakit. Namun, ada perbedaan bermakna keragaman beta pada mikrobiota saluran napas atas antara COVID-19 ringan dan berat. Keberlimpahan filum Firmicutes (p = 0,012), dan genus Streptococcus (p = 0,033) dan Enterococcus (p = 0,031) lebih tinggi pada COVID-19 berat dibandingkan yang ringan, sedangkan keberlimpahan filum Fusobacteriota (p = 0,021), Proteobacteria (p = 0,030), Campilobacterota (p = 0,027), genus Neisseria (p = 0,008), dan Fusobacterium (p = 0,064), spesies Porphyromonas gingivalis (p = 0,018), Fusobacterium periodonticum (p = 0,001) dan Fusobacterium nucleatum (p = 0,022) lebih tinggi pada COVID-19 ringan dibandingkan berat. Keberadaan bakteri Prevotella buccae (p = 0,005) dan Prevotella disiens (p = 0,043) lebih rendah pada COVID-19 berat. Rasio TNF-α/IL-10 lebih tinggi pada COVID-19 berat (p < 0.05). Selanjutnya, rasio IL-6/IL-10, IFN-γ/IL-10, dan IL-1β/IL-10 juga lebih tinggi pada COVID-19 berat, namun tidak berbeda bermakna jika dikaitkan dengan beratnya penyakit. Penelitian ini mendukung adanya hubungan antara karakteristik mikrobiota di saluran napas atas dengan beratnya COVID-19 pada pasien dewasa. Studi lebih lanjut diperlukan untuk memeriksa mekanisme bagaimana mikrobiota mencegah beratnya COVID-19. Rasio TNF-α/IL-10 dari saluran napas dapat menjadi prediktor beratnya penyakit dan sebagai alternatif pemeriksaan kadar sitokin pada COVID-19 yang kurang invasif dibandingkan serum. ......COVID-19 is a disease that caused a pandemic at the end of 2019. Clinical manifestations difference in SARS-CoV-2 infection has raised many questions in research and medical provider. The clinical differences in COVID-19 can be triggered by host, pathogen and environmental factors. SARS-CoV-2 mainly enters through the upper airway, with colonization of commensal and pathogenic microbes. How the interaction between colonized microbes and SARS-CoV-2 in causing an inflammatory response in the upper airway is still not clearly known. Therefore, we examined the association between the diversity of microbiota, pro- and anti-inflammatory cytokines ratio of upper respiratory and COVID-19 severity. This research is an observational cross-sectional study using 74 nasopharyngeal and oropharyngeal swabs in viral transport medium from COVID-19 patients aged 18-64 years. We examined microbiota profile in the upper airway using 16S ribosomal RNA sequencing method and levels of IL-6, IL-1β, IFN-γ, TNF-α and IL-10 were examined by Luminex assay. We also examined the association between COVID-19 severities with OTU analysis, alpha and beta diversity of upper respiratory microbiota. The top five phyla in upper respiratory tract of Indonesian COVID-19 patients with aged of 18–64 years old were Firmicutes (32,3%), Bacteroidota (27,1%), Fusobacteriota (15,2%), Proteobacteria (15,1%) and Actinobacteria (7,1%). Shannon and ACE index analysis showed no decline of microbiota diversity in upper airway with the increase of disease severity. However, there were significant differences of beta diversity in the upper airway microbiota between mild and severe COVID-19. The abundance of the Firmicutes phylum (p = 0,012), Streptococcus (p = 0,033) and Enterococcus (p = 0,031) genera were significantly higher in severe COVID-19 than mild, while the abundance of the Fusobacteriota (p = 0,021), Proteobacteria (p=0,030), and Campilobacterota (p = 0,027) phyla, Neisseria (p = 0,008), and Fusobacterium (p = 0,064) genera, Porphyromonas gingivalis (p = 0,018), Fusobacterium periodonticum (p = 0,001) and Fusobacterium nucleatum (p = 0,022) species were significantly higher in mild. The presence of Prevotella buccae (p=0.005) and Prevotella disiens (p=0.043) bacteria was lower in severe COVID-19. The TNF-α/IL-10 ratio was significantly higher in severe COVID-19 (p < 0.05). Furthermore, IL-6/IL-10, IFN-γ/IL-10, and IL-1β/IL-10 ratio was also higher in severe, but those were not significantly related to the disease severity. This research supports the relationship between the severity of COVID-19 and microbiota diversity in the upper airway in adults. Further studies are needed to examine the mechanism by which microbiota prevents the COVID-19 severities. The ratio of TNF-α/IL-10 from upper airway swab may be as a predictor of disease severity and alternative for examining cytokine levels in COVID-19 which is less invasive than serum.