Previous studies have assessed the stability of flavivirus vaccines using the existing yellow fever and Japanese encephalitis live-attenuated viruses. The unstabilized lyophilized YF 17D vaccine loses 1.5–2.5 log10
/dose (up to 99.7% ) after14 days at 37°C [12
]. A combination of lactose, sorbitol, the divalent cations calcium and magnesium, and at least one amino acid significantly added to the stability of the lyophilized yellow fever vaccine [12
], reducing the vaccine titer loss to less than 1 log after incubation at 37° C for 14 d (U.S. Patent No. 4,500,512). However, this formulation was not effective at preserving DEN-2 PDK-53- candidate vaccine viral titer for 21 hrs at 37°C in the present study. Another study determined that formulations consisting of 10% sucrose alone, 2% sorbitol with 4% inositol, or 10% sucrose with 5% lactalbumin, 0.1g/L CaCl2
and 0.076 g/L MgSO4
provided the best stability for YFV 17D [13
]. However, in all cases after reconstitution, yellow fever vaccine is still very unstable, rapidly losing titer after 2 hrs at 37°C, and must be discarded after only about one hour at room temperature [12
]. This instability leads to vaccine wastage and the potential administration of an ineffective vaccine dose under field conditions. Another live-attenuated flavivirus vaccine against Japanese encephalitis, JEV SA 14-14-2, has been widely distributed in China [14
]. One stabilizing formulation that included 1% gelatin and 5% sorbitol afforded stabilization of the lyophilized vaccine for at least 1.5 yrs at 2–8° C, 4 months at RT, and 10 ds at 37°C. However, the reconstituted liquid vaccine is very labile and its infectious titer decreased significantly after 2 hours at RT [15
]. Although the lyophilized measles mumps and rubella vaccine is stable for months at various temperatures, the reconstituted vaccine loses 2/3 of its infectivity after seven hours at 37°C[16
We have demonstrated improved stability of our live-attenuated flavivirus vaccine candidates with the combination of rHSA, trehalose, and the polyoxyethylene-polyoxypropylene (EO-PO) block copolymer F127. The synergistic effect of these excipients suggests that interactions between these three components and/or between the components and the viral particles contribute to their stabilizing mechanism. The stabilizing role of the albumin in the FTA formulation is not simply as a general carrier protein; other proteins such as gelatin and lactoferrin failed to improve virus stability in our testing (data not shown). We have tested a variety of sources of HSA including recombinant yeast, recombinant rice, and native, human derived proteins, and all provided similar levels of stability for FTA-formulated virus. Use of rHSA would be advantageous, as this would mitigate potential hazards associated with use of human-derived products.
Surfactants have been incorporated into vaccine formulations to prevent material loss to surfaces such as glass vials [17
]. However, commonly used pharmaceutical surfactants such as Polysorbate 20 (Tween 20) were not effective in stabilizing DEN-2 PDK-53, relative to formulations containing a pluronic copolymer (data not shown). Our studies suggested better stabilizing efficiencies with formulations containing distinct, high molecular weight pluronic copolymer surfactants. The pluronic block copolymers have been investigated as potential delivery agents for a variety of hydrophobic drugs, proteins, DNA, or inactivated vaccines [18
]. F127 has also been tested as a carrier for a lentiviral vector to cells of the central nervous system [20
]. Interestingly, non-ionic block copolymers have also been rationally designed to act as adjuvants, and the type of immune response varies by both size and polyolyethylene content [21
]. However, in our studies the FTA formulation does not significantly enhance the immunogenicity of the DEN-2/WN vaccine.
The surfactants studied here display some unusual biochemical properties. Pluronic F127 is a non-ionic polyoxyethylene-poloxypropylene block copolymer, consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. F127 (at concentrations >10%) undergoes a process known as reverse thermogelation, as it undergoes a phase transition from liquid to a gel upon reaching physiological temperatures. Additionally, when these block copolymers reach above the critical concentration they assemble into micelles in aqueous solutions, which is also dependent on temperature [22
]. The critical micelle concentration is 500μM at 20°C, and 2μM at 37°C, respectively [22
]. Under the conditions evaluated here, the FTA formulations have inadequate concentrations to promote reverse thermogelation, but the 1–2% (approximately 800–1600 μM) F127 concentrations tested here permit micelle formation at room temperature and 37°C. The unique biophysical properties of F127 also promote the association of F127 with membranes, and these attributes may result in intercalation of F127 within the membrane of the enveloped flavivirus, promoting its stability.
The studies presented here have successfully identified a combination of pharmaceutically accepted excipients (FTA) which significantly enhances the liquid and freeze-dried thermal stability of candidate DEN-2 PDK-53-based flaviviral vaccines. The excipient components comprising FTA are currently present in approved vaccines, so regulatory issues associated with these components for a live-attenuated vaccine are minimal. We showed that a formulation consisting of 15% trehalose, 1–2% F127 and 0.1–2% human serum albumin synergistically improved the thermal stability of these candidate vaccine viruses. This FTA formulation permitted stable storage of our liquid vaccine for at least 8 hours at 37°C, for 1 week at 25°C, and for at least 11 weeks at 4°C, all with less than 0.5 log titer loss. The formulated vaccine also maintained full viral activity after two freeze/thaw cycles. In addition, FTA formulations were safe in mice and did not affect the immunogenicity of the candidate DEN-2/WN vaccine. Although FTA formulations did not perform better than existing formulations used for stabilization of the liquid yellow fever vaccines, they were effective at stabilizing the DEN-2 PDK-53 and JEV SA 14-14-2 viruses in liquid-phase. Interestingly, YFV17D virus in PBS control was quite stable at 37°C for 21 hours in our experiment, and FTA did not significantly improve the stability (). FTA did not show a stabilizing effect for the liquid YFV 17D vaccine, but did constitute a highly effective stabilizer for DEN-2 PDK-53 virus and its associated DEN-1, DEN-3, DEN-4 and WN chimeras, as well as the JEV SA 14-14-2. In reviewing the phylogeny of the genus Flavivirus
, we hypothesize that the formulation is effective for the dengue virus clade and the Japanese encephalitis virus clade (of which West Nile is a member) but not the yellow fever clade of mosquito-borne flaviviruses [23
We have shown that FTA formulations enhanced thermal stability of the tested live-attenuated vaccine candidates. Based on these data, the formulations appeared to be safe and had no adverse effect on the immunogenic and protective efficacy of the DEN-2/WNV vaccine. In other mouse experiments, FTA did not appear to afford any dose-sparing effect for the candidate DEN-2/WN vaccine (data not shown). Further development of this novel combination of excipients will facilitate distribution of live-attenuated flavivirus vaccines to populations living in areas where maintaining the “cold chain” is difficult. Currently, Inviragen’s DEN-2-PDK-53-based tetravalent dengue vaccine (DENVax), formulated in FTA containing native HSA, is being evaluated for safety and efficacy in Phase 1 clinical trials in healthy adults.