title: Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice.
abstract: Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.
authors.affiliation: Research and Development Centre, Indian Immunologicals Limited, Gachibowli, Hyderabad-500 032, India.
authors.forename: R
authors.initials: R
authors.lastname: Ramya
authors.forename: B
authors.initials: B
authors.lastname: Mohana Subramanian
authors.forename: V
authors.initials: V
authors.lastname: Sivakumar
authors.forename: R L
authors.initials: RL
authors.lastname: Senthilkumar
authors.forename: K R S
authors.initials: KR
authors.lastname: Sambasiva Rao
authors.forename: V A
authors.initials: VA
authors.lastname: Srinivasan
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016367:Baculoviridae; D002021:Buffers; D002460:Cell Line; D003001:Cloning, Molecular; D003902:Detergents; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007313:Insecta; D051379:Mice; D011818:Rabies / Q000276:immunology / Q000517:prevention & control; D011819:Rabies Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D011820:Rabies virus / Q000235:genetics / Q000276:immunology*; D012995:Solubility; D018411:Spodoptera; D016019:Survival Analysis; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D002021:Buffers; D003902:Detergents; D006023:Glycoproteins; D011819:Rabies Vaccines; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus