title: Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.
abstract: Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Chatham Maritime, Kent, UK.
authors.forename: Stuart T
authors.initials: ST
authors.lastname: Mather
authors.affiliation: Viral Pseudotype Unit (Fitzrovia), Faculty of Science and Technology, University of Westminster, London, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Wright
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Chatham Maritime, Kent, UK.
authors.forename: Simon D
authors.initials: SD
authors.lastname: Scott
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Chatham Maritime, Kent, UK. Electronic address: n.temperton@kent.ac.uk.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Temperton
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D005612:Freeze Drying; D057809:HEK293 Cells; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000302:isolation & purification*; D007251:Influenza, Human / Q000276:immunology*; D008379:Marburg Virus Disease / Q000175:diagnosis* / Q000821:virology; D029024:Marburgvirus / Q000276:immunology / Q000302:isolation & purification*; D011818:Rabies / Q000175:diagnosis* / Q000821:virology; D011820:Rabies virus / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral
keywords: Lyophilisation; Neutralising antibodies; Retroviral pseudotype viruses; Serological assays