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Abstrak :
Measles immunization has been introduced since 1960, thereby markedly reducing the number of cases in developed countries. However, measles epidemics still occur even in developed countries. In the United States, in 1988-1992 an increase in the number of measles cases reaching 50,000 cases was reported. Some of these cases occurred in previously immunized patients. This was thought to be caused by genetic mutation of the measles virus, aside from weaknesses of the vaccine and low immunization coverage. Since measles immunization was employed in Indonesia, the number of measles patients has decreased. However, epidemics are still frequently reported. About 15-30% of reported cases occurred in those previously immunized, raising the question of whether a genetic difference exists between the wild-type measles virus circulating in Indonesia and the vaccine virus being used. Such a difference may lead to the differences in the antigenicity of the wild-type and vaccine viruses, rendering the resulting antibody incapable of neutralizing the wild-type viruses. Based on the above, this study is aimed to demonstrate the extent of genetic and antigenic differences between the wild-type and vaccine measles viruses. We conducted an experimental laboratory study to sequence the N, H, and F genes of the wild-type measles viruses (G2, G3, and D9) and the CAM-70 vaccine virus. To show antigenic differences, the wild-type viruses (G2, G3, and D9) and the CAM-70 and Schwarz viruses were injected to BALB/c mice. Serum antibodies of the mice were analyzed using ELISA, cross-neutralization test, and immunoblotting using antigens from the respective viruses. Results of this study showed that the wild-type and the vaccine viruses differ in the sequence of the N gene by 73-79 nucleotides, resulting in amino acid substitution of 17-24 residues; the H gene by 60-99 nucleotides, resulting in amino acid substitution of 13-29 residues; the F gene by 71-88 nucleotides, resulting in amino acid substitution of 4-3 I residues. Differences between the wild-type and the CAM-70 and Schwarz vaccine viruses were also found in the epitope site of the CTL and antibodies, which are important to virus antigenicity. We conclude that a significant difference in antigenicity exists between the wild-type measles viruses circulating in Indonesia with the CAM-70 measles virus. We also found the immunogenicity of the CAM-70 and Schwarz vaccine viruses to be lower than that of the wild-type viruses.

Abstrak :
Dalam upaya menginduksi kekebalan berspektrum luas yang responsif terhadap subtipe-subtipe HIV-1 yang berbeda, telah diteliti imunisasi vaksin DNA menggunakan vektor plasmid DNA dan virus fowlpox rekombinan dengan memanfaatkan gen-gen HIV-1 yang dirancang dari runutan konsensus turunan subtipe-subtipe HIV-1 di dunia yang mengekspresikan semua protein dari genom HIV-1 dengan peptida berukuran 30 asam amino yang overlapping dan tersusun secara acak (scrambled antigen vaccines, atau SAVINE). Tiga grup hewan coba yang terdiri dari masing-masing tujuh beruk (Macaca nemestrina) diimunisasi dengan regimen vaksin DNA standar dengan veklor plasmid DNA pHIS-64 dan vektor virus fowlpox rekombinan (rFPV) berbasis gen gag dan pol dan HIV-1 subtipe B, regimen vaksin DNA SAVINE dengan vektor pHIS-64 dan vektor rFPV berbasis genom HIV-1 yang diacak, serta vektor plasmid pHIS-64 dan FPV yang tidak mengandung gen sebagai grup kontrol. Respon kebal selular diamati dengan teknik ELiSpot dan pewamaan silokin intraselular, sedangkan respon kebal humoral diamati dengan teknik ELISA. Pada ketujuh hewan coba yang diimunisasi dengan vaksin DNA HIV-1 standar, secara umum hasil penelitian menunjukkan terinduksinya respon kebal selular terhadap protein Gag HIV-1 serta respon kebal humoral yang ditunjukkan dengan terdeteksinya antibodi terhadap protein p24 HIV-1. Respon kebal selular silang terhadap protein Gag HIV-1 dari subtipe yang berbeda juga ditunjukkan pada grup yang sama. Namun upaya melakukan imunisasi boosting ke-dua dengan vektor rFPV tidak menunjukkan perbaikan induksi respon kebal. Berbeda dari grup hewan coba yang menerima regimen vaksin DNA HIV-1 standar, pada grup yang menerima regimen vaksin DNA HIV-1 SAVINE secara umum tidak menunjukkan adanya induksi respon kebal, kecuali pada satu ekor hewan yang menunjukkan respon kebal selular yang lebih luas terhadap protein Pol dan protein-protein lain HIV-1 meski pada tingkat induksi yang amat rendah. Pengembangan teknologi vaksin SAVINE terus diperbaiki dan disempumakan dengan kemungkinan melibatkan vektor virus aktif yang lain sehingga induksi respon kebal yang diharapkan bisa tercapai. Specific Immune Responses to the Human Immunodeficiency Virus Type-1 (HIV-1) Proteins In Pigtail Macaque (Macaca nemestrina) Immunized with Whole Gene and Whole Virus Scrambled Antigen Vaccines T cell immunity plays a critical role in controlling HIV-1 viremia, and encoding a limited set of HIV-1 genes within DNA and poxvirus vectors can, when used sequentially, induce high levels of T cell immunity in primates. However, a limited breadth of T cell immunity exposes the host to potential infection with either genetically diverse HIV-1 strains or T cell escape variants of HIV-1. In an attempt to induce maximally broad immunity, we examined DNA (prime) and recombinant Fowlpox virus (rFPV, boost) vaccines encoding all HIV-1 genes derived from a global HIV-1 consensus sequence, but expressed as multiple overlapping scrambled 30 amino acid segments (scrambled antigen vaccines, or SAVINEs). Three groups of 7 pigtail macaques (Macaca nemestrina) were immunized with sets of DNA and rFPV expressing Gag/Pol antigens only, the whole genome SAVINE antigens, or no HIV-1 antigens. T cell immunity was monitored by ELISpot and intracellular cytokine staining, while the humoral immune response was monitored by p24 antibody capture ELISA. High levels of cross-subtype HIV-specific T cell immunity to Gag were consistently induced in the 7 macaques primed with DNA and rFPV vaccines expressing Gag/Poi as intact proteins. The humoral immunity was also induced in the animals from the same group. It was however, difficult to repeatedly boost immunity with further rFPV immunizations, presumably reflecting high levels of anti-FPV immunity. Unfortunately, this vaccine study did not consistently achieve a broadened level of T cell immunity to multiple HIV genes utilizing the novel whole-virus SAVINE approach, with only one of 7 immunized animals generating broad T cell immunity to multiple HIV-1 proteins. Further refinements are planned with alternate vector strategies to evaluate the potential of the SAVINE technology.

Abstrak :
Measles immunization has been introduced since 1960, thereby markedly reducing the number of cases in developed countries. However, measles epidemics still occur even in developed countries. In the United States, in 1988-1992 an increase in the number of measles cases reaching 50,000 cases was reported. Some of these cases occurred in previously immunized patients. This was thought to be caused by genetic mutation of the measles virus, aside from weaknesses of the vaccine and low immunization coverage. Since measles immunization was employed in Indonesia, the number of measles patients has decreased. However, epidemics are still frequently reported. About 15-30% of reported cases occurred in those previously immunized, raising the question of whether a genetic difference exists between the wild-type measles virus circulating in Indonesia and the vaccine virus being used. Such a difference may lead to the differences in the antigenicity of the wild-type and vaccine viruses, rendering the resulting antibody incapable of neutralizing the wild-type viruses. Based on the above, this study is aimed to demonstrate the extent of genetic and antigenic differences between the wild-type and vaccine measles viruses. We conducted an experimental laboratory study to sequence the N, H, and F genes of the wild-type measles viruses (G2, G3, and D9) and the CAM-70 vaccine virus. To show antigenic differences, the wild-type viruses (G2, G3, and D9) and the CAM-70 and Schwarz viruses were injected to BALB/c mice. Serum antibodies of the mice were analyzed using ELISA, cross-neutralization test, and immunoblotting using antigens from the respective viruses. Results of this study showed that the wild-type and the vaccine viruses differ in the sequence of the N gene by 73-79 nucleotides, resulting in amino acid substitution of 17-24 residues; the H gene by 60-99 nucleotides, resulting in amino acid substitution of 13-29 residues; the F gene by 71-88 nucleotides, resulting in amino acid substitution of 4-3 I residues. Differences between the wild-type and the CAM-70 and Schwarz vaccine viruses were also found in the epitope site of the CTL and antibodies, which are important to virus antigenicity. We conclude that a significant difference in antigenicity exists between the wild-type measles viruses circulating in Indonesia with the CAM-70 measles virus. We also found the immunogenicity of the CAM-70 and Schwarz vaccine viruses to be lower than that of the wild-type viruses.

Abstrak :
Latar belakang. Meningkatnya kasus HIV-AIDS human immunodeficiency virus-acquired immunodeficiency syndrome secara global memicu kewaspadaan akan peningkatan infeksi oportunistik, salah satunya infeksi Pneumocystis jirovecii yang mengakibatkan pneumonia PjP. Infeksi PjP merupakan kasus yang sulit ditangani terkait rendahnya sensitivitas uji diagnostik diiringi dengan peningkatan kasus resistensi terhadap antibiotik. Di Indonesia belum terdapat data demografis, epidemiologi molekuler maupun data resistensi mengenai kasus infeksi PjP. Mengantisipasi masalah tersebut, dalam penelitian ini dikembangkan uji diagnostik PjP pada ODHA Orang Dengan HIV-AIDS terduga pneumonia melalui pendekatan molekular terhadap gen MSG Major Surface Glycoprotein disertai dengan karakterisasi gen DHPS dihidropteroat sintase dan gen mtLSU mitochondrial large subunit yang berkorelasi dengan genotipe resisten dan virulensi P. jirovecii. Tujuan penelitian. Memperoleh suatu uji deteksi infeksi PjP, data genotipe resistensi dan virulensi PjP melalui pendekatan secara molekuler yang dapat dimanfaatkan sebagai dasar data demografi dan epidemiologi molekuler PjP di Indonesia. Metode penelitian. Pengembangan uji diagnosis molekuler PjP terhadap gen MSG dilakukan dengan metode real- time PCR yang diujikan terhadap 100 sampel sputum. Pola genotipe resistensi dilakukan melalui amplifikasi gen DHPS dilanjutkan dengan restriction fragment length polymorphism RFLP . Virulensi daerah hot spot gen mtLSU dianalisis dengan metode PCR dan sekuensing DNA. Hasil. Secara demografi, diketahui prevalensi PjP pada ODHA terduga pneumonia di Jakarta mencapai 20,0, laki-laki 75, rentang usia terbanyak 31-40 tahun 35, dominan 80 pada kisaran sel limfosit T CD4 200-349 sel/L. Sebanyak 12 pasien menunjukkan gen DHPS positif, lima pasien 41,66 merupakan genotipe wild type WT dan 7 pasien lainnya 58,32 merupakan genotipe resisten, terdiri dari 16,67 genotipe-3 dan 41,66 genotipe campuran WT dan genotipe 1. Analisis virulensi berdasarkan gen mtLSU diperoleh 30 strain PjP positif yang didominasi oleh variasi-3. Status imun pasien lebih berkaitan dengan genotipe resistensi dibandingkan dengan jenis varian. Kesimpulan. Uji real-time PCR yang dikembangkan mampu memberikan nilai diagnostik yang lebih baik dibandingkan pewarnaan Giemsa. Terdapat 3 genotipe gen resistensi WT, genotipe 1 dan 3 dan 7 varian P. jirovecii yang bersirkulasi di Jakarta. Genotipe resistensi lebih berkaitan terhadap kondisi klinis pasien dibandingkan dengan jenis varian.

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Background. The global rise of HIV-AIDS cases increase the alertness against oportunistic infections, one of them is Pneumocystic jirovecii pneumonia PjP. PjP infection is a one of a tough infection to be cured due to low sensitivity of its diagnostic method following the escalation of PjP resistance against antibiotics. There is no demografic, molecular epidemiology nor antibiotics resistance data were available related to PjP infection in Indonesia. Thus, this study was conducted to develop a molecular test to diagnose PjP infection in HIV-AIDS suspected pneumonia patients based on MSG Major Surface Glycoprotein gene detection, followed by characterization of DHPS dihydropteroat syinthetase and mtLSU mitochondrial large subunit genes represent genoype resistance and P. jirovecii virulence. Research objective. To obtain a molecular test in diagnosing PjP infection and information of P. jirovecii genotype resistance and virulence based on molecular characteristics, which can be used further as demographic and molecular epidemiology basis data of PjP in Indonesia. Research methods. Molecular diagnostic test aimed for MSG gene of P. jirovecii detection was done through real-time PCR against 100 sputum samples. Genotype resistance and P. jirovecii polymorphism patterns was done through DHPS and mtLSU genes amplification followed by restriction fragment length polymorphism RFLP and DNA sequencing analysis. Virulence of the hot spot area are of the mtLSU gene was analyzed by PCR method and DNA sequencing. Results. The prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta was 20.0, male 75 within 31-40 y.o 35, dominant 80 from patients with CD4 T-lymphocytes of 200-349 cells/L. Molecular real-time PCR methods give five times sensitivity higher than Giemsa stain. Twelve patients showed positive DHPS gene, five patients 41.67 were wild type WT genotypes and 7 other patients 58.32 were resistant genotypes, with 16.66 was genotype-3 and other 41.66 was mixed genotypes WT and genotype 1. Virulence analysis based on mtLSU gene show 30 positive strains which dominated by variant-3. The patients immune status is more related to the resistance genotype compared to the variant type. Conclusion. The developed real-time PCR method is proven to able to give better diagnostic value than Giemsa stain. There are 3 genotypes of resistance genes WT, genotypes 1 and 3 and 7 variants of P. jirovecii circulating in Jakarta. Resistance genotypes are more related to the clinical condition of patients compared to variant types.