Best poster award for ITQB PhD student
Rotavirus
Candida Mellado, PhD student at the Animal Cell Technology (ACT) Laboratory, won the best student’s poster award at the international congress Vaccine Technology II. This meeting took place in Albufeira, Algarve, between 1- 6 June, and gathered 170 worldwide participants from academic research institutions, pharmaceutical companies, and world health organizations.
The research work presented in the poster “Novel techniques for characterization of double and triple-layered rotavirus-like particles” is part of the ACT vaccine development strategy. Looking at the assembly mechanisms of virus like particles (VLPs) inside cells, researchers hope to optimize a more cost effective in vitro process for VLP production. VLPs are good candidate vaccines against Rotavirus disease, a disease responsible for the deaths of an estimated 600,000 children each year, 80 percent of whom live in developing countries.
This project results from collaborations between three ITQB/IBET laboratories: Animal Cell Technology, Colloids Polymers and Surfaces, and Pharmacokinetics and Biopharmaceutical Analysis.
Novel techniques for characterization of double and triple-layered rotavirus-like particles
Maria Candida M. Mellado1, Ana Luísa Simplício1, António Lopes1, Manuel J.T. Carrondo1,2, Paula M. Alves1
(1) IBET/ITQB-UNL, Apartado 12, P-2781-901, Oeiras, Portugal
(2) FCT-UNL, Laboratório de Engenharia Bioquímica, P-2825-114, Monte da Caparica, Portugal.
Rotavirus-like particles (RLPs), candidate vaccines against Rotavirus disease, can be produced in insect cells by co-infection of baculoviruses coding for VP2 and VP6 for double-layered RLP 2/6 as well as for VP2, VP6 and VP7 in triple-layered RLP 2/6/7. Analytical methods currently used for virus-like particles (VLPs) characterization include SEC-HPLC, TEM, SDS-PAGE and Western Blot. However, these methodologies are either time-consuming, have little sensitivity, and thus are semi-quantitative or involve a significant amount of sample. Moreover, they do not give information on the particle’s stoichiometry and assembly, which are important issues for improving VLP yield.
In this work novel techniques were applied for RLP 2/6 and RLP 2/6/7 characterization. SDS-capillary gel electrophoresis (SDS-CGE) was successfully used for quantitative and qualitative characterization of RLPs in terms of detecting the presence and relative proportion of VPs for quality control purposes. The method demonstrated to be accurate for apparent molecular weight estimation and allowed evaluation of VP7’s degree of glycosylation. Its precision is sufficient for detection of VP6/VP7 ratio differences between different samples as well as for protein quantification.
Another extremely useful technique to characterize VLPs is dynamic light scattering (DLS), which enables measurement of zeta potential, size and molecular weight. The average diameters of RLP 2/6 and RLP 2/6/7 were 50 and 80 nm, respectively. Several physical-chemical conditions (pH, ionic strength, temperature and chelating agent concentration) were used for size and zeta potential measurement. Besides giving valuable information on RLPs stability and disassembly, DLS also enabled the plot of a titration curve for pI calculation, which was 2.7 (RLP 2/6/7).
Using both methods, stoichiometry, assembly and disassembly kinetics of RLPs are possible to evaluate and quantify.
