title: Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple-layered rotavirus-like particles.
abstract: Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Maria Candida M
authors.initials: MC
authors.lastname: Mellado
authors.forename: Jimmy A
authors.initials: JA
authors.lastname: Mena
authors.forename: António
authors.initials: A
authors.lastname: Lopes
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Laura A
authors.initials: LA
authors.lastname: Palomares
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D012401:Rotavirus / Q000378:metabolism*; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D022701:Virosomes / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D014764:Viral Proteins; D022701:Virosomes