title: Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein.
abstract: Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.
authors.affiliation: Technical Manager, Biomanufacturing Sciences Network, Merck Millipore, Biomanufacturing Sciences and Training Centre, 1 Science Park Road, #02-10/11 The Capricorn, Singapore 117528.
authors.forename: Pradeep B J
authors.initials: PB
authors.lastname: Reddy
authors.forename: Priyabrata
authors.initials: P
authors.lastname: Pattnaik
authors.forename: Nagesh K
authors.initials: NK
authors.lastname: Tripathi
authors.forename: Ambuj
authors.initials: A
authors.lastname: Srivastava
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D053059:Dengue Vaccines / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D003716:Dengue Virus / Q000737:chemistry* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D053059:Dengue Vaccines; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins