Method for lyophilizing live vaccine strains of
There are provided compositions and methods for lyophilization and/or storage of live vaccine strains of Francisella tularensis . More specifically, there are provided lyophilization media and uses thereof for the preparation and long-term storage of Francisella tularensis vaccines.
1. A lyophilization medium for freeze-drying a Francisella tularensis ( F. tularensis ) strain, wherein: the lyophilization medium comprises about 1% (w/v) of mannitol, about 1% (w/v) of a disaccharide, and about 0.25% (w/v) of gelatin in a phosphate buffer; wherein the disaccharide is selected from sucrose, trehalose, and a mixture of sucrose and trehalose; and F. tularensis wherein the F. tularensis strain is a mutant strain in which the clpB gene is inactivated, and wherein initial post-lyophilization viability of the F. tularensis strain is at least about 35% of the F. tularensis strain's pre-lyophilization viability.
2. The lyophilization medium of claim 1 , wherein the medium comprises 1% (w/v) mannitol, 1% (w/v) sucrose, and 0.25% (w/v) gelatin in 10 mM phosphate buffer pH 7.2.
3. The lyophilization medium of claim 1 , wherein the disaccharide is sucrose.
4. A method for lyophilizing a live vaccine strain of Francisella tularensis ( F. tularensis ) comprising the steps of: 1) pelleting a culture of the live vaccine strain by centrifugation; 2) resuspending the pelletized live vaccine strain in the lyophilization medium of claim 1 to form a suspension of the live vaccine strain in the lyophilization medium; and 3) freeze-drying the suspension.
5. A method for lyophilizing a live vaccine strain of Francisella tularensis ( F. tularensis ) suspended in a growth medium comprising the steps of: 1) replacing the growth medium with the lyophilization medium of claim 1 to obtain a suspension of the live vaccine strain in the lyophilization medium; and 2) freeze-drying the suspension.
6. The method of claim 5 , wherein the growth medium is replaced with the lyophilization medium using filtration.
7. The method of claim 1 , wherein the mutant strain is derived from a wild-type clinical strain of F. tularensis selected from the group consisting of SCHU S4, FSC033, FSC108, and FSC200.
8. The method of claim 6 , wherein the filtration is tangential flow filtration.
9. A method for lyophilizing a live vaccine strain of Francisella tularensis ( F. tularensis ) comprising the steps of:
1. thawing a concentrated frozen liquid preparation of the live vaccine strain comprising the lyophilization medium of claim 1 ; and
2. freeze-drying the thawed preparation.
10. A method for lyophilizing a live vaccine strain of Francisella tularensis ( F. tularensis ) comprising the steps of:
1. suspending the live vaccine strain in the lyophilization medium of claim 3 ; and
2. freeze-drying the suspension.
11. A method for lyophilizing a live vaccine strain of Francisella tularensis ( F. tularensis ) comprising the steps of:
1. suspending the live vaccine strain in the lyophilization medium of claim 4 ; and
2. freeze-drying the suspension.
12. A composition comprising a lyophilization medium and a strain of Francisella tularensis ( F. tularensis ), wherein the lyophilization medium comprises about 1% (w/v) of mannitol, about 1% (w/v) of a disaccharide, and about 0.25% (w/v) of gelatin in a phosphate buffer; and wherein the disaccharide is selected from sucrose, trehalose, and a mixture of sucrose and trehalose and wherein initial post-lyophilization viability of the F. tularensis strain is at least about 35% of the F. tularensis strain's pre-lyophilization viability.
13. A lyophilizate of the composition of claim 12 .