Protective Efficacy of Lyophilized Vesicular Stomatitis Virus–Based Vaccines in Animal Model

L ive recombinant vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (VSV∆G/EBOVGP) was evaluated during 2014-2015 as a vaccine to limit the effects of EBOV disease (1).Because of the success and safety of the EBOV vaccine, similar VSV-based vaccines have been proposed for Sudan and Marburg viruses and for other etiologic agents of viral hemorrhagic fever diseases, such as Lassa virus (LASV) (2).Cold chain maintenance for distributing and storing VSV-based vaccines is a logistical challenge, especially when ultralow temperatures (-60°C to -80°C) are required.The challenge is greater in rural areas, particularly in developing countries, where infrastructure and transport systems are often deficient.We evaluated the effects of lyophilization on the in vitro recoverability and in vivo protective efficacy of VSV-based vaccines.We conducted animal studies in accordance with the Canadian Council of Animal Care guidelines; studies received approval from the Canadian Science Centre for Human and Animal Health's institutional Animal Care and Use Committee.We performed work involving infectious Lassa virus in a Biosafety Level 4 laboratory within the Public Health Agency of Canada.When required, we inactivated materials for subsequent analysis according to approved procedures. The StudyWe conducted propagation and titration (50% tissue culture infectious dose [TCID 50 ]) of VSV∆G/EBOVGP and VSV-based LASV (VSV∆G/LASVGPC) vaccines by using Vero E6 cells as previously described (3).We evaluated 4 excipients as stabilizers: 2.5% lactalbumin hydrolysate (L), 5% sucrose (S), 2.5% trehalose (T), and 0.25% gelatin (G).We prepared 2× concentrations of each solution initially in Hanks' balanced salt solution and then evaluated 3 combinations (LS, LST, or LSTG) (4,5).The control formulation for lyophilization was Dulbecco modified Eagle medium (DMEM) without additives.We mixed each excipient combination 1:1 with VSV∆G/EBOVGP (stock titer 1.26 × 10 7 TCID 50 /mL) or VSV∆G/LASVGPC (2.83 × 10 7 TCID 50 /mL) and dispensed 200 µL of the mixture into 4 mL sterile glass vials (Electron Microscopy Sciences, https://www.emsdiasum.com).We lyophilized the vaccine mixtures by using an automated FreeZone Triad Benchtop Freeze Dryer (Labconco, https:// www.labconco.com)according to the manufacturer's specifications (Appendix Table, https://wwwnc.cdc.gov/EID/article/30/5/23-1248-App1.pdf).We stored the vials at 4°C, 21°C, or 37°C for 1, 7, 30, and 90 days after lyophilization.At those time points, we reconstituted each vaccine/stabilizer combination in triplicate in 200 µL of 0.85% saline for 1 hour at room temperature by using gentle agitation.We then prepared 10-fold serial dilutions in DMEM and determined virus titers by using standard TCID 50 methodologies, as previously described (3).Titrations of formulations conducted immediately before lyophilization indicated that the addition of stabilizers had Protective Efficacy of Lyophilized Vesicular Stomatitis Virus-Based Vaccines in Animal Model

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