engleski

Application of monolithic columns for murine and human cytomegalovirus purification

The impact of human CMV (HCMV) infections on public health is significant while affecting the most vulnerable groups, immunocompromised individuals and congenitally infected infants. Thus, a vaccine to reduce the incidence and severity of HCMV infection is a public health priority. Moreover, cytomegalovirus has a number of features that makes it a very interesting vector platform for gene therapy. In both cases, preparation of highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the best studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. The transfer of knowledge from MCMV and mice to HCMV and humans could face challenges not only because of differences in the immune systems of these two species, but also because of differences in the biological and biophysical properties of the two viruses. We investigated possibilities to purify both viruses by using monolithic chromatography. We also emphasized the purity and functionality of viral particles, as they ensure the safety and strength of the vaccine. The effectiveness of the procedures was monitored using CCID50 assay, Nanoparticle tracking analysis (NTA), ELISA for host cell proteins, and quantitative PCR assay for host cell DNA. Ion-exchange chromatography showed that both, HCMV and MCMV, bind to anion exchange column and not to the cation exchange column, meaning that the HCMV and MCMV particles are negatively charged. HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support, while the recovery of MCMV particles was around 75 %. During HCMV purification process most of the host cell proteins were found in the flow through fraction, and most of host cell DNA eluted after the peak with most of the infective HCMV particles. As for the exosomes, they showed the same behaviour as HCMV. These results provide important data for research on all downstream processes on these two viruses regarding biotechnological production and basic research.

cytomegalovirus, HCMV, MCMV, ion-exchange chromatography, virus

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