Characterization of New RSV-F-Mutants Using BAC Technology Reflecting Their Impact on Viral Growth and Palivizumab Susceptibility

Abstract

Respiratory syncytial virus (RSV) is one of the most serious pathogens causing severe lower respiratory infections in young children, in the elderly and immunocompromised patients. Globally, there were as many as 118 200 deaths of children under 5 years caused by RSV in 2015. Despite clear clinical needs, treatment approaches for severe RSV-infection remain unsatisfactory. Palivizumab is a monoclonal antibody that targets the fusion F protein on the RSV envelope and is prophylactically administered to high-risk children to prevent a severe RSV infection. Several palivizumab resistance-associated mutations in the RSV-F protein were identified and characterized. Marker transfer analysis is a method that allows a reliable characterization of newly detected mutations with unknown phenotype, and for RSV it is just available to a limited extent. This work engaged itself with the establishment of maker transfer analysis for RSV and standardization of in vitro assays characterizing identified mutations in the RSV-F gene with respect to their influence on viral replication and palivizumab susceptibility. Via “en passant” mutagenesis, mutations were respectively introduced into the bacterial artificial chromosome (BAC) pSynkRSV-l19F (BEI Resources Nr-36460). Generation of recombinant RSV-F-Mutants was successful and stable within this system. Growth of recombinant virus and palivizumab susceptibility were then examined in vitro. In this work, nine single mutations were characterized: C21G, Q34R, R49K, T100S, A103P, K272E, N276S, A518V, C550Y, whereby K272E and N276S are known mutations. Furthermore, the following combinations of mutations C21G/R49K, Q34R/C550Y and T100S/A518V were characterized. Recombinant RS viruses do not differ from wildtype virus in their growth kinetics. Concerning palivizumab susceptibility, it can be confirmed that mutation K272E confers to the virus a stable resistance to palivizumab. Mutations R49K and A103P resulted in 2-fold higher IC50 values compared to the parental strain. RSV variants with mutation Q34R or C550Y were similarly susceptible to palivizumab as the parental strain. Remarkably, mutant that contained both mutations Q34R and C550Y showed significant higher IC50 value compared to the reference strain. This might point out that accumulation of trivial mutations might impact the viral phenotype and thus mutations should be characterized in the context of the whole genetic background. In addition, a new thickening agent, colloidal microcrystalline cellulose was tested for replacement of methylcellulose in the overlay medium used in plaque reduction neutralization assay (PRNA). A concentration of 1.5% colloidal microcrystalline cellulose was shown to be sufficient for the use in PRNA.