Production- and Purification-Relevant Properties of Human and Murine Cytomegalovirus.

Sanda Ravlić,Lea Hiršl,Marija Brgles,Beata Halassy,Stipan Jonjić

doi:10.3390/v13122481

Abstract

There is a large unmet need for a prophylactic vaccine against human cytomegalovirus (HCMV) to combat the ubiquitous infection that is ongoing with this pathogen. A vaccination against HCMV could protect immunocompromised patients and prevent birth defects caused by congenital HCMV infections. Moreover, cytomegalovirus (CMV) has a number of features that make it a very interesting vector platform for gene therapy. In both cases, preparation of a highly purified virus is a prerequisite for safe and effective application. Murine CMV (MCMV) is by far the most studied model for HCMV infections with regard to the principles that govern the immune surveillance of CMVs. Knowledge transfer from MCMV and mice to HCMV and humans could be facilitated by better understanding and characterization of the biological and biophysical properties of both viruses. We carried out a detailed investigation of HCMV and MCMV growth kinetics as well as stability under the influence of clarification and different storage conditions. Further, we investigated the possibilities to concentrate and purify both viruses by ultracentrifugation and ion-exchange chromatography. Defective enveloped particles were not separately analyzed; however, the behavior of exosomes was examined during all experiments. The effectiveness of procedures was monitored using CCID50 assay, Nanoparticle tracking analysis, ELISA for host cell proteins, and quantitative PCR for host cell DNA. MCMV generally proved to be more robust in handling. Despite its greater sensitivity, HCMV was efficiently (100% recovery) purified and concentrated by anion-exchange chromatography using QA monolithic support. The majority of the host genomic DNA as well as most of the host cell proteins were removed by this procedure.

Highlights

Cytomegaloviruses (CMVs) are enveloped dsDNA prototypes of the β subfamily of Herpesviridae
Our studies showed that Human CMV (HCMV) grows more slowly on MRC-5 cells than Murine CMV (MCMV) on M210B4 cells and that the highest HCMV infectivity can be expected after the eighth day, post infection with all investigated multiplicity of infection (MOI)
A good feature of HCMV growth on MRC-5 cells is that the virus can be repeatedly harvested to yield higher amounts of infective particles, as shown in Figure 1A, which is not the case with MCMV growth on M2-10B4 cells because the cytopathic effect strongly destroys the cells

Summary

Introduction

Cytomegaloviruses (CMVs) are enveloped dsDNA prototypes of the β subfamily of Herpesviridae. There is a strong interest in CMV as an engineered vector for vaccination against other viral diseases and as “live drugs”, including therapeutic oncolytic agents [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30] This great attention is based on the size and engineering flexibility of the CMV genome [23] along with its superinfection capacity [5], which goes beyond pre-existing immunity [24]. There are no licensed CMV vaccines and the publications on their pharmaceutical development are limited; great efforts are being made to better understand the virus [33]

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