Mezenkimal Kök Hücreler Üzerinde Dengue Virüsüne Karşı (DENV-1,2,3,4) Fab IgG Hibrit Antikorunun Nötralize Edici Potansiyeli
Dengue humması, tropik ve subtropikal ülkelerde sivrisineklerden kaynaklanan akut viral bir enfeksiyon olup, her yıl giderek artmaktadır. Endonezya’daki Dang kanamalı ateşine DENV-1, DENV-2, DENV-3 ve DENV-4 adı verilen 4 viral serotip neden olmaktadır. Hastalığın tedavisi çok zordur ve Dang virüsü enfeksiyonlarını yok etmek için etkili bir aşı yoktur. Bu araştırmanın amacı, sıçan kemik iliği mezenkimal kök hücrelerinde (sıçan BM-MSCs) bulunan Fab IgG antikorunun VL ve VH antikor fragmentini kodlayan geninin açığa çıkarılmasıdır. Genler, tüm dang serotiplerinin inaktif virüsü kullanılarak aşılanmış farelerden izole edilmiştir. Sonrasında genler, ligasyon ve plasmid pBR322 içine gömülmesi yoluyla hibridize edilmiş ve sıçan BMMSC’lerine nakledilmiştir. Analitik olarak Fab IgG’nin hibrid bağlama yeteneği, kapasitesi ve özgünlüğünü karakterize etmek için; bağışıklık çökeltmesi, western blotting, nötralizasyon testi ve ELISA kullanılmıştır. Bütün deneyler, hibrid Fab IgG antikorunun tüm Dang virüsü serotipleri 1,2,3,4’ü etkili bir şekilde nötrleştirmek için yüksek reaktiviteye ve afiniteye sahip olduğunu ortaya koymaktadır. Bu çalışmanın sonuçları, melez Fab IgG antikorunun gelecekte dang enfeksiyonlarının tedavisinde nötralize edici ajan olarak kullanılabileceğini göstermektedir.
Neutralizing Potential of Fab IgG Hybrid Antibody Against Dengue Virus (DENV-1,2,3,4) Expressed on Mesenchymal Stem Cells
Dengue is an acute mosquito born viral infections in tropicaland subtropical countries, that has been steadily increasingevery year. Dengue haemorrhagic fever in Indonesia is causedby all 4 viral serotypes termed as DENV-1, DENV-2, DENV-3,and DENV-4. It is very difficult to treat the disease and there isno effective vaccine to eradicate Dengue virus infections .Theaim of this research is to express the antibody fragment genecoding VL and VH of Fab IgG antibody in rat bone marrow mesenchymal stem cells (rat BM-MSCs). The genes were isolatedfrom immunized mice using inactive virus of all dengue serotypes. Then genes were hybridized through ligation and insertion into plasmid pBR322, and transfected into rat BMMSCs.To analytically characterize Fab IgG hybrid binding ability, capacity and specificity, we used immune precipitation, westernblotting, neutralization assay, and ELISA. All assays suggestedthat hybrid Fab IgG antibody have high reactivity and affinity,to efficiently neutralize all Dengue virus serotypes 1,2,3,4. Results of this study show that hybrid Fab IgG antibody can beused as neutralizing agent for the treatment of dengue infections in the future.
___
- Abaitua F., Rodríguez, J.R., Garzón, A., Rodríguez, D., Esteban, M.
2006. Improving recombinant MVA immune responses: potentiation of the immune responses to HIV-1 with MVA and DNA vectors
expressing Env and the cytokines IL-12 and IFNgamma. Virus Res
116, 11-20. [CrossRef]
- Bielefeldt-Ohmann, H., Meyer, M., Fitzpatrick, D.R., Mackenzie, J.S.
2001. Dengue virus binding to human leukocyte cell lines: receptor
usage differs between cell types and virus strains. Virus Res 73(1), 81–9.
[CrossRef]
- Champion, J.M., Kean, R.B., Rupprecht, C.E., Notkins, A.L., Koprowski, H., Dietzschold, B., Hooper, D.C. 2000. The development of monoclonal human rabies virus-neutralizing antibodies as
a substitute for pooled human immune globulin in the prophylactic
treatment of rabies virus exposure. J. Immunol. Methods 235, 81–90.
[CrossRef]
- Chu., Y.K., Rossy, C., LeDuc, J.W., Lee, H.W., Schmaljohn, C.S., Dalrymple. 1994. Serological relationship among virus in the hanta virus
genus family bunyaviridae virology 198, 196-204
- Crameri, R., 2007. Allergy vaccines: dreams and reality. Expert Rev Vaccines 2007 Dec; 6(6), 991-9. [CrossRef]
- de Carvalho Nicacio, C., Lundkvist, A., Sjölander, K.B., Plyusnin, A.,
Salonen, E.M,. Björling, E. 2000. A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus. J Med Virol
60, 446-454. [CrossRef]
- Fried, J.R., Gibbons, R.V., Kalayanarooj, S., Thomas, S.J., Srikiatkhachorn, A., Yoon, I.K., Jarman, R.G., Green, S., Rothman, A.L.,
Cummings, D.A. 2010. Serotype-specific differences in the risk of
dengue hemorrhagic fever: an analysis of data collected in Bangkok, Thailand from 1994 to 2006. PLoS Negl Trop Dis 4(3), e617.
[CrossRef]
- Gigler A., Dorsch, S., Hemauer, A., Williams, C., Kim, S., Young,N.S.,
Zolla-Pazner, S., Wolf, H., Gorny M.K., Modrow, S. 1999.
Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins. J Virol 73, 1974-1979.
- Gould, L.H., Sui, J,. Foellmer, H., Oliphant, T., Wang, T., Ledizet, M.,
Murakami, A., Noonan, K., Lambeth, C., Kar, K., Anderson, J.F.,
de Silva, A.M., Diamond, M.S., Koski, R.A., Marasco, W.A., Fikrig,
E. 2005. Protective and therapeutic capacity of human single-chain
Fv-Fc fusion proteins against West Nile virus. J Virol 79, 14606-14613.
[CrossRef]
- Halstead, S.B. 1998. Pathogenesis of dengue: challenges to molecular
biology. Science 239, 476-481. [CrossRef]
- Halstead, S.B. 2003. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 60, 421-467. [CrossRef]
- Henchal, E.A., Putnak, J.R. 1990. Dengue viruses. Clin Microbiol Rev
3, 376-396. [CrossRef]
- Huse, K., Böhme, H.J., Scholz, G.H. 2002. Purification of antibodies
by affinity chromatography. Journal of Biochemical and Biophysical
Methods 51(3), 217-231. [CrossRef]
- Kehry, M.R., Castle, B.E. 1994. Regulation of CD40 ligand expression
and use of recombinantCD40 ligand for studyingBcell growth and
differentiation. SeminImmunol 6, 287-294. [CrossRef]
- Kihira, Y., Aiba, S. 1992. Artificial immunoglobulin G-binding protein
mimetic to staphylococcal protein A. Its production and application
to purification of immunoglobulin G. J Chromatogr B: Biomed Sci
Appl 597, 277-283.
- Lai, C.Y., Tsai, W.Y., Lin, S.R., Kao, C.L., Hu, H.P., King, C.C., Wu, H.C.,
Chang, G.J., Wang, W.K. 2008. Antibodies to envelope glycoprotein
of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol 82(13), 6631-
6643. [CrossRef]
- Leickt, L., Grubb, A., Ohlson, S. 1998. Screening for weak monoclonal
antibodies in hybridoma technology. J Mol Recognit 11(1-6), 114-
116. [CrossRef]
- Liang, W.Q., Fournier, M.J. 1997. Synthesis of functional eukaryotic
ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis. Proc Natl Acad Sci USA 94,
2864-2868. [CrossRef]
- Meulenbroek, A.J., Zeijlemaker, W.P. 1996. Human IgG subclasses:
useful diagnostic markers for immunocompetence. Amsterdam CLB,
1-51.
- Ong, S.H., Yip, J.T., Chen, Y.L., Liu, W., Harun, S., Lystiyaningsih, E.,
Heriyanto, B., Beckett, C.G., Mitchell, W.P., Hibberd, M.L., Suwandono, A., Vasudevan, S.G., Schreiber, M.J. 2008. Periodic re-emergence of endemic strains with strong epidemic potential-a proposed
explanation for the 2004 Indonesian dengue epidemic. Infect Genet
Evol 8(2), 191-204. [CrossRef]
- Pauli, G., Gregersen, J-P., Ludwig, H. 1984. Plaque/ focus immunoassay: a simple method for detecting antiviral monoclonal or other antibodies and viral antigens in cell. J Immunol Methods 74, 337-344.
[CrossRef]
- Putnak, R., Fuller, J., Vander Zanden, L., Innis, B.L., Vaughn, D.W.
2003. Vaccination of rhesus macaques against dengue-2 virus with
a plasmid DNA vaccine encoding the viral pre-membrane and envelope genes. Am J Trop Med Hyg 68(4), 469-476.
- Rantam, F.A., Purwati, Soegijanto, S., Susilowati, H., Sudiana, K.,
Hendrianto, E., Soetijipto. 2013. Analysis of recombinant, multivalent dengue virus containing envelope (E) proteins from serotypes
- 1, - 3, and - 4 and expressed in baculovirus. Trials Vaccinol DOI:
10.1016/j.trivac.2013.10.001. [CrossRef]
- Rantam, F.A., Ferdiansyah, Nasronudin, Purwati. 2009. Stem Cell
Exploration. Airlangga University Press, Surabaya, 70-75.
- Rothman, A.L. 2010. Cellular immunology of sequential dengue virus
infection and its role in disease pathogenesis. Curr Top Microbiol Immunol 338, 83-98. [CrossRef]
- Sarathy, V.V., White, M., Li, L., Gorder, S.R., Pyles, R.B., Campbell,
G.A., Milligan, G.N., Bourne, N., Barrett, A.D. 2015. A lethal murine infection model for dengue virus 3 in AG129 mice deficient in
type I and II interferon receptors leads to systemic disease. J Virol
89(2), 1254-1266. [CrossRef]
- Sui, J., Li, W., Roberts, A., Matthews, L.J., Murakami, A., Vogel, L.,
Wong, S.K., Subbarao, K., Farzan, M., Marasco, W.A. 2005. Evaluation of human monoclonal antibody 80R for immunoprophylaxis of
severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants. J Virol 79, 5900-5906. [CrossRef]
- Sumarmo, 1987. Dengue haemorrhagic fever in Indonesia. Southeast
Asian J Trop Med Public Health 18, 269-274.
Tagliabue, A., Rappuoli, R. 2008. Vaccine adjuvants: the dream becomes real. Hum Vaccine 4, 347-349. [CrossRef]
- Wu, S.J., Grouard-Vogel, G., Sun, W., Mascola, J.R., Brachtel, E.,
Putvatana, R., Louder, M.K., Filgueira, L., Marovich, M.A., Wong,
H.K., Blauvelt, A., Murphy, G.S., Robb, M.L., Innes, B.L., Birx, D.L.,
Hayes, C.G,. Frankel, S.S. 2000. Human skin Langerhans cells are
targets of dengue virus infection. Nat Med 6(7), 816-820. [CrossRef]
- Yamanaka, A., Mulyatno, K.C., Susilowati, H., Hendrianto, E., Ginting, A.P., Sary, D.D., Rantam, F.A., Soegijanto, S., Konishi, E. 2011.
Displacement of the predominant dengue virus from type 2 to type
1 with a subsequent genotype shift from IV to I in Surabaya, Indonesia 2008–2010. PLoS One 6, e27322. [CrossRef]
- Yokoyama, T., Ayabe, S., Miyagi, H., Sugano, T., Otsu, A., Sato, H.,
Kageyama, S., Fujii, T., Shiraki, K. 2001. Varicella-zoster virus gH:gL
contains a structure reactive with the anti-human gamma chain of
IgG near the glycosylation site. J Gen Virol 82, 331-334. [CrossRef]
- Yoshida, T., Yoshida, R., Ma, B.Y., Mikolajczak, S., Kelvin, D.J., Ochi.
A. 2010. A novel mitogen fusion protein against CD40+ cells with
potent vaccine adjuvant properties. Vaccine 28(21), 3688-3695.
[CrossRef]