Untersuchungen zum Einfluss veränderter Aminosäuresequenzen im Haemagglutinin-Neuraminidase-Protein aktueller Mumpswildviren im Vergleich zum Impfvirus

Abstract

The Mumps virus belongs to the family of paramyxoviridea and is a single-stranded, negative-sensed RNA virus. The genome encodes for nine proteins. The focus of this study was on the hemagglutinin-neuraminidase protein (HN-protein) which is essential in virus neutralizing. In the last years increasing reports of mumps outbreaks in vaccinated or naturally infected populations were documented. Most outbreaks belonged to the genotype G whereas genotype A is mostly used for vaccination. Some results of former studies suggested that antibodies formed by vaccine strains neutralize wild-type viruses less effectively, leading to discussions concerning the development of a new mumps vaccine. Recent studies of the “Robert Koch-Institute” showed that a third dose of mumps-containing vaccine does not increase protection and also waning immunity was ruled out as a factor of reinfection. The aim of this study was to investigate the impact of natural occurring mutations in the HN-protein on the binding capacity of sera from vaccinated or naturally infected individuals. For this purpose, six constructs of mutated HN have been generated before. These constructs are based on amino acid alterations in regions already known or suspected to be relevant for neutralization. As the binding capacity of human antibodies to HN was supposed to be tested in an Immunofluorescence Assay (IFA), first of all staining conditions with human sera had to be established. Solitary expressed HN could neither be detected with human sera using various conditions nor with mouse monoclonal antibodies (kindly provided by C. Örvell) which were used as a control. To determine whether the detection of HN by IFA requires viral context, MuV infected Vero cells were stained with human sera or the monoclonal antibodies. Five monoclonal antibodies were able to detect HN in infected cells while the human sera still failed to detect the HN-protein. That was a surprising result because IFA is a method often used in diagnostic surveys and serum titer could determined by ELISA, a method based on a similar principle as IFA. Another research group, which used the same plasmid construct encountered the same problem. It might be possible that transfected HN is not correctly folded or modified (e.g. not properly glycosylated) due to the N-terminal myc-epitop of the used plasmid. Although the impact of natural occuring mutations could not be determined in this study, the results can be used for serological diagnostics and studies using plasmid constructs with myc-epitopes.