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

Heri Setiyo Bekti, Silvia Tri Widyaningtyas, Budiman Bella

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.

 

 

 

 


Keywords


Gag and Vp22-Gag recombinant proteins; intracellular migration, Gag-specific IgG response.

References


Stephen GP, David KM, Peter HM, editors. Retroviridae. In fields virology. 7th ed. Philadelphia: Lippincott Williams & Wilkins; 2013.

Campbell EM, Hope TJ. HIV-1 capsid: the multifaceted key player in HIV-1 infection. Nat Rev Micro. 2015 Agustus; 13: 471-83.

Delves JP, Martin JS, Burton RD, Roitt MI. Immunology: Roitt’s essential. 12th ed. West Sunsex: John Wiley & Sons Ltd; 2015.

Wang HB, Mo QH, Yang Z. HIV vaccine research: the challenge and the way forward. J Immunol Res. 2015;503978.

Kementrian Kesehatan R.I. Situasi dan analisa HIV-AIDS [serial on the internet]. 2014 [cited 2016 Januari 5]. Available from: http://www.depkes.go.id/resources/download/pusdatin/infodatin/pdf.

Valentina M, Natasha P, Marianne H, Linda A, Julio MSG. Adverse effects of antiretroviral therapy for HIV infection. CMJ. 2004;170(2):229-38.

Dilernia DA, Mónaco DC, Krolewiecki A, César C, Cahn P, Salomón H. The importance of early diagnosis for the survival of HIV positive patients. Medicina. 2010;70(5):453-6.

Barré-Sinoussi F, Ross AL, Delfraissy JF. Past, present and future: 30 years of HIV research. Nat Rev Micro. 2013 Dec;11:877-83.

Taylor D, Durigon M, Davis H, Archibald C, Konrad B, Coombs D, et al. Probability of a false negative HIV antibody test result during the window period: a tool for pre- and post-test counselling. Int J STD AIDS. 2014;1–10.

World Health Organization. Draft global health sector strategy on HIV, 2016-2021 [serial on the internet]. 2015 [cited 2016 Januari 5]. Available from: http://www.who.int/hiv/pub/hiv_strategy/en/.

Cohen ZY, Raphael D. Novel HIV vaccine strategies: overview and perspective. Ther Adv Vaccines. 2013; 13(1):99-112.

Coico R, Sunshine G. Immunology: A short course. 7th ed. West Sunsex: John Wiley & Sons Ltd; 2015.

Kevin AK, Guido V, Eric AJ. Is HIV-1 evolving to a less virulent form in humans?. Nat Rev Micro. Feb 2007;5:141-49.

Ashwini SV, Madhuri TR, Srikanth TT, Raur CG, Sekhar C, Ramesh PS. T-cell epitope indentified by BALB/c mice immunized with vaccinia expressing HIV-1 Gag lie within immunodominant regions recognized by HIV-infected Indian patients. jgid. 2011;3(3): 246-53.

Graham BS, Wrigit PF. Drug therapy: Candidate AIDS Vaccines. NEJM. 2015;333(20): 1331-39.

Gupta S, Termini JM, Raffa FN, Williams CA, Kombluth RS, Stone GW. Vaccination with a fusion protein that introduces HIV-1 Gag antigens into a multitrimer CD40L construct results in enhanced CD8+ T Cell responses and protection from viral challenge by vaccinia - Gag. J. Virol. 2014;88(3):1492-1501.

Briggs JAG, Riches JD, Glass B, Bartonova V, Zanetti G, Krausslich HG. Structure and assembly of immature HIV. PNAS. 2009;106(27)11090-95.

Luis BA, Derek OT. Designing and bluiding the next generation of improved vaccine adjuvan. JCR. 2014;190:563-79.

Sukumar S, Shinsuk Y, Kenji O, Kiyohiko M, Tomoko M, Fumihiko T, et al. A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus. J. Virol; 2006:48–57.

Kelly H, Sue-Li D, Lucy PX, Tobias C, Par N. VP22 core domain from Herpes simplex virus 1 reveals a surprising structural conservation in both the Alpha- and Gammaherpesvirinae subfamilies. J Gen Vir. 2015;96:1436-45.

Michiko T, Akihisa T, Yuko S, Takahiro I, Ken S, Kayo K, et al. Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes Neurovirulence. J. Virol. 2012;86(9):5264-77.

Nishikawa M, Otsuki T, Ota A, Guan X, Takemoto S, Takahashi Y, et al. Induction of tumor-spesific immune response by gene transfer of Hsp70-cell-penetrating peptide fusion protein to tumor in mice. J Mol Ther. 2010;18(2):421-28.

Christopher S, Jurgen H, Ajoy SK, Schulze-Osthoff K. Specific inhibition of transcription factor NF-κB through intracellular protein delivery of IκBα by the Herpes virus protein VP22. Oncogene. 2003;22:5367-73.

Gillian E, Peter O. Interceluler trafficking and protein delivery by a Herpesvirus structural protein. Cell Press. 1997;88:223-33.

Xian Y, Zhengmin X, Jun L, Ting Ting L, Yan W. VP22 mediates intercellular trafficking and enhances the in vitro antitumor activity of PTEN. Mol Med Rep. 2015;12: 1286-90.

Karine S, Laurent F, Carole S, Lee L, Patrick M. CD4 T cell help is required for primary CD8 T cell responses to vesicular antigens delivered to dendritic cells in vivo. Eur. J. Immunol. 2006;36:1386-97.

Mehrad M, Rita CJ, Rafi A. CD4+ T cells are required to sustain CD8+ T cell responses during chronic viral infection. J. Virol.1994;68(12):8056-63.

PPLVKP FK UI-RSCM. Standar opersional prosedur laboratorium Biomedik. Jakarta; 2013.

Qiagen. The QIA Expressionist: A handbook for high-level expression and purification of 6xHis-tagged proteins. 5th ed. [serial on the internet]. 2003 Jun [cited 2016 Januari 5]. Available from: http:// kirschner. med. harvard.edu /files/protocols /QIAGEN_QIAexpressionist_EN.pdf

Sumarno EP, Chandra KHMS, Istiadji ESM, Darto S. Adrenocorticotrophin hormone 4-10 synthetic inhibit prostaglandin E2 and IL-1β in LPS-induce meningitis. ijpsi. 2013;2(3):1-5.

Silvia WT. Pengembangan penghantaran DNA berbasis Polipetida sebagai upaya meningkatkan efisiensi dan efektifitas transfeksi DNA pada sel mamalia [Desertasi]. Jakarta: Universitas Indonesia; 2015. Indonesian.

George K, Anna-Katharina S, Frank W, Wai-Ling K, Ling-Pei H, David S, et al. Potent adaptive immune responses induced against HIV-1 gp 140 and influenza virus HA by a polyanionic carbomer. Vaccine. 2010; 28: 2482-89.

Silvia C, Cinzia F, Eleonora G, Federica D, Egidia CB, Antonella C, et al. Identification of new HIV-1 gag-specific cytotoxic T lymphocyte responses in BALB/c mice. JVirol. 2008;81(5):1-7.

Sopiyudin DM. Statistik untuk Kedokteran dan Kesehatan. Jakarta: Salemba Medika; 2016. Indonesian.

Marit K, Ivan S, Stephen KJ. The role of HIV-specific antibody-dependent cellular cytotoxicity in HIV prevention and the influence of the HIV-1 Vpu protein. AIDS. 2014; 29:137–44.

Briand L, Marcion G, Kriznik A, Heyde M, Artur Y, Garrido C, et al. A self-inducible heterologous protein expression system in Escherichia coli [serial on the internet]. 2016 [cited 2017 February 12]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017159/.

Anja M, Bettenbrock K. Lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. Els J Biotech. 2012;157:82–88.

German RL, Eduardo CA. Recombinant protein expression in Escherichia coli: advances and challenges. 2014;5(172):1-17.

Sivashanmugam, Victoria M, Chunxian C, Yonghong Z, Jianjun W, Qiangin L. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Scie. 2009;18:936-48.

Joanne C, Brigitte MS, Joachim R. One-Step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin. Spring Mol Biothec.1995;4:247-58.

Suvra R, Vikash K. A Practical Approach on SDS PAGE for Separation of Protein. IJSR. 2012;3(8):955-60.

BioRad. Electrophoresis: a guide to Polyacrylamide gel electrophoresis and detection [serial on the internet]. 2014 [cited 2017 February 12]. Available from: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf.

Zahid A, Jamil W, Begum R. Method development and validation of SDS-PAGE for quality control testing of Pegylated interferon Alpha-2a. IOSR-JPBS. 2014;9(6):32-6.

Arianna R, Fiona C, Charles DM. Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts. PNAS. 2013;110(39).

Joshua BA, Joseph FJ. Purification of Proteins using Polyhistidine affinity tags. PubMed. 2000;326:245-54.


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