Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays

Purpose: Filoviruses, such as Ebolavirus, are zoonotic pathogens causing disease outbreaks with high mortality rates, requiring scarce high containment facilities for research. Nevertheless, pseudotyped viruses (PV), consisting of a lentiviral core (plus luciferase reporter) and the envelope glycoprotein (GP), allow basic and translational virology to be conducted under low containment. Consequently, filovirus PVs were generated and viability assessed after lyophilisation and long-term storage. Next, antibody neutralisation tests were performed using native and hybrid GPs to assess differentiation between genera and species.

Methods & Materials: PVs were produced using a 3-plasmid transfection system (representing core, reporter and envelope) in HEK293T/17 cells, and supernatant titrated. Supernatants were then lyophilised in sucrose cryoprotectant solution, stored under various conditions, reconstituted and titrated. For antibody neutralisation tests, serially diluted, polyclonal convalescent sera (NIBSC, UK) or anti-GP monoclonal antibodies (Xiangguo Qiu, PHA, Canada; Erica Saphire, Scripps, USA) were incubated with PV for 1 h at 37 °C, prior to titration. To create artificial GP antigens, EBOV neutralising epitopes were inserted into the GP of another genus (Cuevavirus; LLOV) by mutagenesis, PVs generated and infectivity and neutralisation assessed.

Results: High titre PVs were produced with titres between ∼1 × 108 RLU/mL (Ebolavirus/Cuevavirus)and ∼1 × 1010 RLU/mL (Marburgvirus).

Lyophilised PV titres remained constant stored at −20 °C and 4 °C for 12 months, while PVs kept at room temperature (22.5 °C) demonstrated titre decreases of up to 3 orders of magnitude after 6 months. At 37 °C, five log (Marburgvirus) or three log (Ebolavirus and Cuevavirus) decreases occurred after one month.

Zaire Ebolavirus (EBOV) antibodies showed no cross reactivity with native LLOV PVs. Furthermore, EBOV epitopes inserted into the LLOV GP and expressed on PVs had no significant impact on PV infectivity, and EBOV neutralising epitopes were successfully reconstituted in these chimeric antigens

Conclusion: In this study, high titre PVs were generated and found to be amenable to lyophilisation and long-term storage. Reconstituted PVs retained their function in neutralisation assays suggesting their structure is not compromised during freeze-drying. Insertion of epitopes in heterologous GPs did not impact infectivity or functionality. This data suggests a PV-based serological kit could be utilised in resource-limited countries for serological studies, after simple refrigeration storage.