Quantitative determination of the surfactant-induced split ratio of influenza virus by fluorescence spectroscopy

Abstract

The majority of marketed seasonal influenza vaccines are prepared using viruses that are chemically inactivated and treated with a surfactant. Treating with surfactants has important consequences: it produces 'split viruses' by solubilizing viral membranes, stabilizes free membrane proteins and ensures a low level of reactogenicity while retaining high vaccine potency. The formulation stability and potency of split influenza vaccines are largely determined by the specifics of this 'splitting' process; namely, the consequent conformational changes of proteins and interactions of solubilized particles, which may form aggregates. Robust methods to quantitatively determine the split ratio need to be developed before optimal splitting conditions can be investigated to streamline production of superior influenza vaccines.Here, we present a quantitative method, based on both steady-state and time-resolved fluorescence spectroscopy, to calculate the split ratio of the virus after surfactant treatment. We use the lipophilic dye Nile Red (NR) as a probe to elucidate molecular interactions and track changes in molecular environments. Inactivated whole influenza viruses obtained from a sucrose gradient were incubated with NR and subsequently treated with increasing concentrations of the surfactant Triton X-100 (TX-100) to induce virus splitting. NR's emission spectra showed that the addition of TX-100 caused~27 nm red-shifts in the emission peak, indicative of increasingly hydrophilic environments surrounding NR. The emission spectra of NR at different surfactant concentrations were analyzed with multi-peak fitting to ascertain the number of different micro-environments surrounding NR and track its population change in these different environments. Results from both the emission spectra and fluorescence lifetime spectroscopy revealed that NR showed presence in 3 distinct molecular environments. The split ratio of the virus was then calculated from the percentages of NR in these environments using both fluorescence emission and lifetime data. This study can pave the way for the development of robust methods to rapidly quantify splitting extent during vaccine manufacturing. KEYWORDS fluorescence emission; fluorescence lifetime; influenza split virus vaccine; influenza virus; membranesurfactant interactions; Nile Red; partitioning of dye in membranes; protein aggregation; split ratio quantification; surfactant; Triton X-100; vaccine formulation; vaccine manufacturing; virus splitting; virus-surfactant interactions

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“…The strong correlation between solution pH and structural stability of the virus is a proof-of-concept for our methodology to stabilize the rabies vaccine. Recently we reported the effect of a surfactant on the influenza virus with observations akin to this study, albeit with different excipients and conditions (27). Our current method allows preparing the vaccine in many different buffers (e.g., Tris or potassium phosphate) and can also be applied in other lyophilized vaccines or therapeutic protein formulations where pH is controlled by CO 2 or other methods in the liquid form but is possible to be shifted upon lyophilization.…”

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“…However, they lose some of the inherent immunogenicity because of the lack of the whole virus structure, unlike WIV [ 87 ]. The split-virus vaccine can be prepared by disrupting the virus membrane chemically using surfactants such as Triton-100 or Octyl glycoside [ 89 ]. Then, the surfactant is removed by tangential flow filtration.…”

“…These aggregates can be caused by removing the splitting agent (surfactant) in the manufacturing of split-virus influenza vaccines [ 180 ]. In addition to EM and light scattering, UV−Vis absorption spectroscopy and fluorescence emission spectroscopy are reliable techniques in identifying protein aggregations [ 89 , 172 ]. EM is a direct imaging tool for determining the size and shape of large proteins or a complex of proteins.…”