Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt^®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac^®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac^®, and demonstrated that the DuckCelt^®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac^® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt^®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt^®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac^®.

Since the early 2000’, regulation agencies have encouraged viral vaccine manufacturers to implement in-process and real-time monitoring tools in production processes. Even if more assays have been recently developed, none of the novel viral particle quantification technologies can monitor virus levels and their secretion kinetics within production vessels. Vaccine manufacturers still rely on offline cell-based infectivity assays and antigen amount quantification to monitor their processes. The present study describes the development of the first automated biosensor for at-line monitoring of influenza virus production. It involves coupling a fetuin-based SPRi quantitative biosensor with an automated sampler of culture broth and a consecutive clarification setup via an acoustic filter. The SPRi response of different viral strains produced in two distinct cell production platforms was qualified. We demonstrated that fetuin-based quantitative SPRi is a robust, potency-indicating, and universal analytical technology for quantifying bioactive influenza virus particles. It was validated with both purified and complex matrices. Finally, an influenza viral production kinetic was monitored online for three days. This novel online tool enabled the access in real-time to total bioactive viral particles from early production phases (8hpi).

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen for which there is currently no specific treatment or licensed vaccine. Different strategies have been evaluated to prevent this infection, including the use of live-attenuated vaccines (LAVs). However, further development of LAV approaches is often hampered by the lack of highly efficient and scalable cell-based production systems that support worldwide vaccine production. In this context, avian cell lines cultivated in suspension are currently competing with traditional cell platforms used for viral vaccine manufacturing. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel) we previously described as an efficient production system for several influenza strains could also be used to produce a new HMPV LAV candidate (Metavac®), an engineered SH gene-deleted mutant of the A1/C-85473 strain of HMPV. To that end, we characterized the operational parameters of multiplicity of infection (MOI), cell density, and trypsin addition to achieve optimal production of the LAV Metavac® in the DuckCelt®-T17 cell line platform. We demonstrated that the DuckCelt®-T17 cell line is permissive and is well adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, as well as its capacity to induce efficient neutralizing antibodies in a mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.