HIV-1 Gag specific IgG response in mice immunized with Vp22-Gag vaccine candidate

Abstract

Latar Belakang: Stimulasi respon sel T CD8+ spesifik Gag, terkait dengan penurunan viremia, kontrol replikasi virus, dan perkembangan penyakit yang lambat. Respon T CD8+ yang efektif juga dipengaruhi oleh sel T CD4+. Protein rekombinan Gag dapat diklona dan diekpresikan pada sistem prokariota, dan pada saat diimunisasi pada hewan coba atau manusia akan bersifat sebagai antigen eksogen. Antigen eksogen dapat menjadi antigen endogen dengan menambahkan protein yang mempunyai kemampuan bertranslokasi kedalam membran sel, salah satunya protein Vp22.Metode: Transformasi Plasmid rekombinan didapatkan dari Pusat Penelitian dan Layanan Virologi Kanker Patobiologi Fakultas Kedokteran Universitas Indonesia-Rumah Sakit Pusat Nasional dr. Cipto Mangunkusumo (PPLVKP FK UI-RSCM), yang dilakukan transformasi pada sistem ekpresi prokariota dengan metode heat shock, yang dilanjutkan dengan ekpresi protein rekombinan. Purifikasi protein rekombinan dilakukan dengan kromatografi afinitas. Analisa berat molekul protein rekombinan dilakukan dengan SDS-PAGE. Western blotting dilakukan untuk mengaetahui reaktifitas protein rekombinan dengan antibodi poliklonal terhadap antigen p24. Transfeksi sel CHO dan imunisasi mencit DDY dengan protein rekombinan, untuk mengetahui kemampuan migrasi intraseluler serta stimulasi respon imun spesifik.Hasil: Uji western blotting, menunjukkan protein rekombinan dapat berinteraksi dengan antibodi poliklonal terhadap antigen p24. Pengamatan mikroskop konfokal menunjukkan protein rekombinan berlokalisasi dengan endosom. Uji ELISA, menunjukkan respon IgG spesifik Gag setelah imunisasi pada mencit DDY. Kesimpulan: Protein rekombinan dapat diekspresikan pada sistem ekspresi prokariota. Kemampuan migrasi intrasseluler protein rekombinan pada sel CHO belum dapat dibuktikan. Protein rekombinan dapat menstimulasi respon IgG spesifik Gag. Kata Kunci: Protein rekombinan Gag dan Vp22-Gag; Migrasi intraseluler, Respon IgG spesifik Gag. Abstract Background: Stimulation of Gag-specific CD8+ T-cell response, associated with reduction in viremia, viral replication control, and slow disease progression. Effective CD8+ T cell response is also influenced by CD4+ T cells. Gag recombinant protein may be cloned and expressed in the prokaryotic system and when they are immunized in experimental animals or human will have property as exogenous antigens. Exogenous antigens may become endogenous antigens by adding proteins that have the ability to translocate into the cell membrane, one of which is the Vp22 protein. Methods: Recombinant plasmids were obtained from Research and Services Centers of Virology and Cancer Patobiology Medical Faculity Universitas Indonesia-dr. Cipto Mangunkusumo National Central General Hospital (PPLVKP FK UI-RSCM) which transformation to prokaryotic expression system with heat shock method was followed by expression of recombinant proteins. Purification of recombinant proteins was performed with affinity chromatography. The molecular weight analysis of recombinant proteins was performed with SDS-PAGE. Western blotting was performed to determine the reactivity of recombinant proteins with polyclonal antibodies against p24 antigens. Transfection of Chinese Hamster Ovary (CHO) cell and immunization of Deutschland, Denken, and Yoken (DDY) mice with recombinant proteins was conducted to determine intracellular migration ability and stimulation of specific immune response. Results: Western blotting test, indicating recombinant protein may interact with polyclonal antibody against p24 antigens. The observation of a confocal microscope showed recombinant proteins localized with endosomes. The Enzyme-linked Immunosorbent Assay (ELISA) test indicates Gag-specific IgG response after immunization in DDY mice.Conclusion: Recombinant proteins may be expressed on a prokaryotic expression system. The ability of recombinant protein intracellular migration in CHO cell has not been proven. Recombinant proteins may stimulate Gag-specific IgG response. Keywords: Gag and Vp22-Gag recombinant proteins; intracellular migration, Gag-specific IgG response.