Compositions and methods for freeze-drying bacteria or Listeria strains

Abstract

To provide methods and compositions for the lyophilization of a bacterium or a Listeria strain, e.g., Listeria monocytogenes.SOLUTION: Provided are: a method for producing a lyophilized composition comprising a bacterium or a Listeria strain; a formulation for lyophilization containing a bacterium or a Listeria strain; a lyophilized bacterium or Listeria strain; and a method for preparing a frozen bacterium or Listeria strain for lyophilization. Some such methods may comprise: providing a composition comprising a bacterium or a Listeria strain in a formulation comprising a buffer; cooling the composition in a freezing step; exposing the cooled composition to a vacuum and a first temperature increase in a primary drying step; and exposing the composition from the primary drying step to a vacuum and a second temperature increase in a second drying step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 560,318, filed September 19, 2017, which is incorporated herein by reference in its entirety for all purposes.

[0002] Reference to sequence listings submitted as text files via EFS Web The sequence listing set forth in file 519152SEQLIST.txt is 89.2 kilobytes, was created on August 24, 2018, and is hereby incorporated by reference. [Background technology]

[0003] background Lyophilization is a process of removing solvent from a solution to form a solid or powder that is more stable than a liquid and easier to store at high temperatures. Lyophilization, also known as freeze-drying, involves freezing followed by sublimation. The resulting lyophilized material can be stored without freezing or at higher temperatures than a liquid, thereby reducing the cost of storing and transporting materials and the storage space required for the product. Lyophilization can also reduce the weight of the product, thus reducing shipping and related costs. Lyophilization is particularly useful for preserving and storing various biomolecules because it extends their shelf life.

[0004] Compared with liquid formulations, solid formulations have several advantages, such as excellent storage stability, reduced molecular mobility and undesired chemical reactions, and lower packaging weight for easier shipping and distribution. Furthermore, because all currently available commercial vaccines require low-temperature storage, the goal is to utilize solid-state stabilization technology to increase their stability at room temperature or higher and reduce their dependence on the cold chain to maintain efficacy and ensure safety. The low storage and transportation costs of freeze-dried bacterial cultures are a major advantage compared to cryopreservation, making lyophilization a preferred preservation method. However, lyophilization is a highly complex physical process affected by many parameters that require specialized equipment and trained personnel. Freeze-drying can cause many types of damage to cells, including reduced viability, reduced metabolic activity, and altered cell morphology, which can affect the physiology, properties, and functions of cells such as bacteria.

[0005] Additionally, the effects of various different freeze-drying parameters are highly strain-specific, and this strain dependence makes it difficult to draw general conclusions or guidance from any one particular strain. Due to the high biological and metabolic diversity of bacteria, developing bacteria-specific optimized freeze-drying procedures is difficult and arduous. There is very limited data regarding the freeze-drying of Listeria strains, such as Listeria monocytogenes, as well as which parameters need to be optimized to make freeze-drying a viable option for Listeria, and how to optimize those parameters. Summary of the Invention [Means for solving the problem]

[0006] Abstract Methods and compositions are provided for freeze-drying bacteria or Listeria strains, such as Listeria monocytogenes. In one aspect, a method for producing a freeze-dried composition comprising bacteria or Listeria strains is provided. Some such methods can include providing a composition comprising bacteria or Listeria strains in a formulation comprising a buffer, cooling the composition in a freezing step, exposing the cooled composition to a vacuum and a first elevated temperature in a primary drying step, and exposing the composition from the primary drying step to a vacuum and a second elevated temperature in a secondary drying step, thereby producing the freeze-dried composition.

[0007] In some such methods, the bacteria or Listeria strains used in the composition are frozen Listeria strains that are thawed prior to the freezing step. In particular examples, the frozen bacteria or Listeria strains can be thawed at a temperature of about 2°C to about 37°C, about 20°C to about 37°C, about 23°C to about 37°C, about 25°C to about 37°C, about 32°C to about 37°C, or about 37°C. Optionally, thawing is for about 8 hours or less. Optionally, the thawed bacteria or Listeria strains are held at a temperature of about 2°C to about 8°C for about 24 hours or less. In particular examples, the concentration of the thawed bacteria or Listeria strains is about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 It can be between colony forming units (CFU).

[0008] In some such methods, the formulation comprises a buffer and sucrose. For example, the formulation buffer may comprise about 1% to about 5% w / v sucrose, about 2% to about 3% w / v sucrose, or about 2.5% w / v sucrose. Optionally, the formulation does not contain one or more other excipients, such as trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA).

[0009] In some such methods, the formulation contains about 1 x 10 per milliliter9 ~Approx. 1×10 10 Contains colony forming units (CFU) of bacteria or Listeria.

[0010] In some such methods, the holding temperature in the primary drying step is between about -10°C and about -30°C, between about -12°C and about -22°C, between about -17°C and about -19°C, or about -18°C.

[0011] In some such methods, the residual moisture in the lyophilized composition is at least about 2.5%, at least about 3%, or at least about 3.5%. In some such methods, the residual moisture is between about 1% and about 5%, or between about 2% and about 4%.

[0012] In some such methods, the lyophilized composition exhibits a viability of at least about 60%, 70%, 80%, or 90% after storage between about -20°C and about 4°C, or after storage at about -20°C or about 4°C for about 6 months, 12 months, 18 months, or 24 months.

[0013] Such methods can include, for example, the steps of: (a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose; (b) cooling the composition provided in step (a) to a holding temperature of between about −32° C. and about −80° C. in a freezing step; (c) exposing the composition produced by step (b) to a vacuum at a holding temperature of between about −10° C. and about −30° C. in a primary drying step; and (d) exposing the composition produced by step (c) to a vacuum at a holding temperature of between about −5° C. and about 25° C. in a secondary drying step, thereby producing a lyophilized composition. Alternatively, such a method can include, for example, the steps of: (a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose; (b) cooling the composition provided in step (a) to a holding temperature of between about −32° C. and about −80° C. in a freezing step; (c) exposing the composition produced by step (b) to a vacuum at a holding temperature of between about −10° C. and about −30° C. in a primary drying step; and (d) exposing the composition produced by step (c) to a vacuum at a holding temperature of between about 5° C. and about 25° C. in a secondary drying step, thereby producing a freeze-dried composition. In some such methods, the Listeria strain is a recombinant Listeria monocytogenes strain, a stress response is induced in the Listeria strain by exposing the Listeria strain to a reduced temperature, the buffer is a phosphate buffer, the formulation comprises 2% to 3% w / v sucrose, the formulation does not comprise trehalose, MSG, or rHSA, the temperature in the primary drying step (c) is between -17°C and -19°C, and the residual moisture in the lyophilized composition is between 3% and 4%. Some such methods have one or more or all of the following elements: the Listeria strain is a recombinant Listeria monocytogenes strain; the buffer is a phosphate buffer; the formulation comprises about 2% to about 3% w / v sucrose; the formulation does not comprise trehalose, MSG, or rHSA; and the formulation contains about 1 x 10 per milliliter. 9 ~Approx. 1×10 10the holding temperature in the primary drying step (c) is between about -17°C and about -19°C; the holding temperature in the secondary drying step (d) is between -1°C and 1°C; and the residual moisture in the freeze-dried composition is between about 2.5% and about 4%. In some such methods, the Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a). Optionally, such methods have one or more or all of the following elements: the concentration of the thawed frozen Listeria strain is about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 The freeze-drying method is a freeze-drying method for freeze-drying Listeria strains. ...

[0014] In another aspect, a formulation for lyophilization containing bacteria or Listeria strains is provided. Such formulations may include, for example, (1) a Listeria strain, (2) a phosphate buffer, and (3) sucrose. In some such formulations, the Listeria strain is a recombinant Listeria monocytogenes strain, the formulation contains about 2% to about 3% w / v sucrose, and the formulation does not contain trehalose, MSG, or rHSA.

[0015] In another embodiment, a lyophilized composition comprising a bacterium or Listeria strain is provided. Some such lyophilized compositions have a residual moisture content of at least about 2.5% or at least about 3%. Some such lyophilized compositions may further comprise a phosphate buffer and sucrose. In some such lyophilized compositions, the Listeria strain is a recombinant Listeria monocytogenes strain, the lyophilized composition does not contain trehalose, MSG, or rHSA, and the residual moisture content in the lyophilized composition is between 3% and 4%.

[0016] In another aspect, methods are provided for preparing frozen Listeria strains for lyophilization, comprising thawing the frozen Listeria strains at a temperature between about 20° C. and about 37° C. Optionally, such methods have one or more or all of the following elements: the thawed frozen Listeria strain has a concentration of about 1×10 per milliliter; 9 ~Approx. 1×10 10 The colony forming units (CFU) are: frozen Listeria strains thawed at about 37°C; frozen Listeria strains thawed for no more than 8 hours; and frozen Listeria strains maintained at about 2°C to about 8°C for no more than 24 hours after thawing. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for producing a lyophilized composition comprising a Listeria strain, comprising: (a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose; (b) cooling the composition provided in step (a) to a holding temperature of between about −32° C. and about −80° C. in a freezing step; (c) exposing the composition produced by step (b) to a vacuum in a primary drying step at a holding temperature of between about −10° C. and about −30° C.; and (d) exposing the composition produced by step (c) to a vacuum at a holding temperature of between about −5° C. and about 25° C. in a secondary drying step. thereby producing a lyophilized composition. (Item 2) 2. The method of claim 1, wherein prior to step (a), a stress response is induced in the Listeria strain by exposing the Listeria strain to a reduced temperature. (Item 3) 2. The method of claim 1, wherein prior to step (a), a stress response is not induced in the Listeria strain by exposing the Listeria strain to a reduced temperature. (Item 4) The method of any preceding claim, wherein the Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a). (Item 5) The frozen Listeria strain is thawed to a concentration of about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 The method according to item 4, wherein the number of colony-forming units (CFU) is between 1 and 2. (Item 6) 6. The method of claim 4 or 5, wherein the frozen Listeria strain is thawed at about 2°C to about 37°C. (Item 7) 7. The method of claim 6, wherein the frozen Listeria strain is thawed at about 20°C to about 37°C. (Item 8) 8. The method of claim 7, wherein the frozen Listeria strains are thawed at about 32°C and about 37°C. (Item 9) Item 10. The method of item 8, wherein the frozen Listeria strain is thawed at about 37°C. 10. The method of any one of items 4 to 9, wherein the frozen Listeria strain is thawed for 8 hours or less. (Item 11) 11. The method according to any one of items 4 to 10, wherein the frozen Listeria strain is kept at about 2°C to about 8°C for 24 hours or less after thawing. (Item 12) 4. The method of any one of items 1 to 3, wherein the Listeria strain used in the composition in step (a) is freshly cultured prior to step (a). (Item 13) The method according to any preceding item, wherein the buffer solution is a phosphate buffer solution. (Item 14) The method according to any preceding item, wherein the formulation comprises about 1% to about 5% w / v sucrose. (Item 15) 15. The method of claim 14, wherein the formulation comprises about 2% to about 3% w / v sucrose. (Item 16) 16. The method of claim 15, wherein the formulation comprises about 2.5% w / v sucrose. (Item 17) The formulation contains about 1 x 10 per milliliter 9 ~Approx. 1×10 10 The method of any preceding claim, comprising colony forming units (CFU) of Listeria. (Item 18) The method of any preceding item, wherein the formulation does not include one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). (Item 19) 19. The method of claim 18, wherein the formulation does not contain trehalose, MSG, or rHSA. (Item 20) The method according to any one of the preceding items, wherein the holding temperature in the freezing step (b) is between about -40°C and about -50°C. (Item 21) 21. The method of claim 20, wherein the holding temperature in the freezing step (b) is about −45° C. (Item 22) 10. The method of claim 1, wherein the freezing step (b) comprises decreasing the temperature to the holding temperature at a rate of about 1° C. per minute. (Item 23) The method according to any one of the preceding items, wherein the cooling in the freezing step (b) is for about 2 hours to about 4 hours. (Item 24) 2. The method of claim 1, wherein the cooling in the freezing step (b) comprises holding the composition at the holding temperature for about 2 hours. (Item 25) The method according to any one of the preceding items, wherein the holding temperature in the primary drying step (c) is between about -12°C and about -22°C. (Item 26) Item 26. The method according to item 25, wherein the holding temperature in the primary drying step (c) is between about -17°C and about -19°C. (Item 27) 27. The method of claim 26, wherein the holding temperature in the primary drying step (c) is about −18° C. (Item 28) 10. The method of claim 1, wherein the primary drying step (c) comprises increasing the temperature to the holding temperature at a rate of about 1° C. per minute. (Item 29) The method according to any one of the preceding items, wherein the primary drying step (c) is performed for about 25 hours to about 35 hours. (Item 30) The method according to any of the preceding items, wherein the primary drying step (c) is completed about 12 to about 16 hours after the composition reaches the holding temperature. (Item 31) The method of any preceding claim, wherein the primary drying step (c) is at a vacuum pressure of about 0.09 mbar. (Item 32) The method according to any one of the preceding items, wherein the holding temperature in the secondary drying step (d) is between about -5°C and about 20°C. (Item 33) Item 33. The method according to item 32, wherein the holding temperature in the secondary drying step (d) is between about -5°C and about 5°C. (Item 34) Item 34. The method of claim 33, wherein the holding temperature in the secondary drying step (d) is about 0°C. (Item 35) The method of any preceding claim, wherein the secondary drying step (d) comprises increasing the temperature to the holding temperature at a rate of about 0.2°C per minute. (Item 36) The method according to any one of the preceding items, wherein the secondary drying step (d) is performed for about 1 hour to about 10 hours. (Item 37) The method according to any one of the preceding items, wherein the secondary drying step (d) comprises holding the composition at the holding temperature for about 2 hours to about 6 hours. (Item 38) Item 38. The method of claim 37, wherein the secondary drying step (d) comprises holding the composition at the holding temperature for about 5 hours to about 6 hours. (Item 39) The method of any preceding claim, wherein the secondary drying step (d) is at a vacuum pressure of about 0.09 mbar. (Item 40) The method according to any of the preceding items, wherein the residual moisture in the freeze-dried composition is between about 1% and about 5%. (Item 41) Item 41. The method of item 40, wherein the residual moisture in the lyophilized composition is between about 2% and about 4%. (Item 42) 10. The method of any preceding claim, wherein the residual moisture in the lyophilized composition is at least about 2.5%. (Item 43) 43. The method of claim 42, wherein the residual moisture in the lyophilized composition is at least about 3%. (Item 44) The method according to any of the preceding items, wherein the freeze-dried composition exhibits a viability of at least about 60% after storage at about -20°C to about 4°C for about 12 months. (Item 45) Item 45. The method according to item 44, wherein the freeze-dried composition exhibits a viability of at least about 75% after storage at about -20°C to about 4°C for about 12 months. (Item 46) Item 46. The method according to item 45, wherein the freeze-dried composition exhibits a viability of at least about 80% after storage at about -20°C to about 4°C for about 12 months. (Item 47) 10. The method of any preceding claim, wherein the Listeria strain is a recombinant Listeria monocytogenes strain. (Item 48) the Listeria strain is a recombinant Listeria monocytogenes strain; the buffer solution is a phosphate buffer solution, the formulation comprises about 2% to about 3% w / v sucrose; the formulation does not contain trehalose, MSG, or rHSA; The formulation contains about 1 x 10 per milliliter 9 ~Approx. 1×10 10 Contains colony-forming units (CFU) of Listeria the holding temperature in the freezing step (a) is between about −40° C. and about −50° C.; the holding temperature in the primary drying step (c) is between −17° C. and −19° C.; the holding temperature in the secondary drying step (d) is between -1°C and 1°C; the residual moisture in the lyophilized composition is between about 2.5% and about 4%; The method according to any of the preceding items. (Item 49) the Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a); The frozen Listeria strain is thawed to a concentration of about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 Between colony forming units (CFU), the frozen Listeria strains are thawed at about 37°C; the frozen Listeria strains are thawed for 8 hours or less; The frozen Listeria strain is kept at about 2°C to about 8°C for 24 hours or less after thawing. Item 49. The method according to item 48. (Item 50) 10. The method of any preceding claim, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, the fusion polypeptide comprising a PEST-containing peptide fused to a disease-associated antigenic peptide. (Item 51) 51. The method of claim 50, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein. (Item 52) 51. The method of claim 50, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO. (Item 53) A formulation for lyophilization of a Listeria strain, comprising: (1) a Listeria strain; (2) a phosphate buffer; and (3) sucrose. (Item 54) 54. The formulation of item 53, wherein the Listeria strain is a strain in which a stress response has been induced by exposing the Listeria strain to a reduced temperature. (Item 55) 55. The formulation of item 53 or 54, wherein the Listeria strain is from a frozen Listeria stock. (Item 56) 55. The formulation of item 53 or 54, wherein the Listeria strain is from a freshly cultured Listeria stock. (Item 57) 57. The formulation of any one of items 53 to 56, comprising about 1% to about 5% w / v sucrose. (Item 58) 58. The formulation of item 57, comprising about 2% to about 3% w / v sucrose. (Item 59) 59. The formulation of item 58, comprising about 2.5% w / v sucrose. (Item 60) 60. The formulation of any one of items 53 to 59, which does not contain one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). (Item 61) 61. The formulation of item 60, which does not contain trehalose, MSG, or rHSA. (Item 62) 62. The formulation of any one of items 53 to 61, wherein the Listeria strain is a recombinant Listeria monocytogenes strain. (Item 63) the Listeria strain is a recombinant Listeria monocytogenes strain; the formulation comprises about 2% to about 3% w / v sucrose; The formulation does not contain trehalose, MSG, or rHSA. The formulation according to any one of items 53 to 62. (Item 64) 64. The formulation of any one of items 53 to 63, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide. (Item 65) 65. The formulation of item 64, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein. (Item 66) 65. The formulation of item 64, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO. (Item 67) A freeze-dried composition produced by the method according to any one of Items 1 to 52. (Item 68) A lyophilized composition comprising a Listeria strain, a phosphate buffer, and sucrose. (Item 69) 69. The lyophilized composition of item 68, which does not contain one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). (Item 70) 70. The lyophilized composition of item 69, which does not contain trehalose, MSG, or rHSA. (Item 71) 71. The freeze-dried composition according to any one of items 67 to 70, wherein the residual moisture in the freeze-dried composition is between about 1% and about 5%. (Item 72) 72. The freeze-dried composition of claim 71, wherein the residual moisture in the freeze-dried composition is between about 2% and about 4%. (Item 73) 73. The freeze-dried composition according to any one of items 67 to 72, wherein the residual moisture in the freeze-dried composition is at least about 2.5%. (Item 74) 74. The freeze-dried composition of claim 73, wherein the residual moisture in the freeze-dried composition is at least about 3%. (Item 75) A freeze-dried composition comprising a Listeria strain, wherein the residual moisture in the freeze-dried composition is at least about 2.5%. (Item 76) 76. The freeze-dried composition of any one of items 67 to 75, exhibiting a viability of at least about 60% after storage at between about -20°C and about 4°C for about 12 months. (Item 77) 77. The freeze-dried composition of item 76, exhibiting at least about 75% viability after storage between about -20°C and about 4°C for about 12 months. (Item 78) 78. The freeze-dried composition of item 77, exhibiting at least about 80% viability after storage at about -20°C to about 4°C for about 12 months. (Item 79) 79. The freeze-dried composition of any one of items 67 to 78, wherein the Listeria strain is a recombinant Listeria monocytogenes strain. (Item 80) the Listeria strain is a recombinant Listeria monocytogenes strain; the lyophilized composition does not contain trehalose, MSG, or rHSA; The residual moisture in the lyophilized composition is between 2.5% and 4%. 80. The freeze-dried composition according to any one of items 67 to 79. (Item 81) 81. The lyophilized composition of any one of items 67 to 80, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, the fusion polypeptide comprising a PEST-containing peptide fused to a disease-associated antigenic peptide. (Item 82) 82. The lyophilized composition of claim 81, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein. (Item 83) 82. The lyophilized composition of claim 81, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO. (Item 84) 1. A method of preparing frozen Listeria strains for lyophilization, comprising thawing the frozen Listeria strains at a temperature between about 20°C and about 37°C. (Item 85) Item 85. The method according to item 84, wherein the temperature is between about 32°C and about 37°C. (Item 86) 86. The method of claim 85, wherein the temperature is about 37°C. (Item 87) 87. The method of any one of items 84 to 86, wherein the frozen Listeria strain is thawed for 8 hours or less. (Item 88) 88. The method of any one of items 84 to 87, wherein the frozen Listeria strain is kept at about 2°C to about 8°C for no more than 24 hours after thawing. (Item 89) 89. The method of any one of items 84 to 88, wherein the frozen Listeria strain is thawed in a formulation comprising a buffer and sucrose. (Item 90) 90. The method of claim 89, wherein the formulation comprises about 1% to about 5% w / v sucrose. (Item 91) 91. The method of claim 90, wherein the formulation comprises about 2% to about 3% w / v sucrose. (Item 92) 92. The method of claim 91, wherein the formulation comprises about 2.5% w / v sucrose. (Item 93) 93. The method of any one of items 89-92, wherein the formulation does not comprise one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). (Item 94) 94. The method of claim 93, wherein the formulation does not contain trehalose, MSG, or rHSA. (Item 95) 95. The method of any one of items 89 to 94, wherein the Listeria strain is a recombinant Listeria monocytogenes strain. (Item 96) the Listeria strain is a recombinant Listeria monocytogenes strain; the formulation comprises about 2% to about 3% w / v sucrose; The formulation does not contain trehalose, MSG, or rHSA. 96. The method according to any one of items 89 to 95. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows viable cell count (VCC) data over time for different formulation buffers (citrate, phosphate, and MOPS (3-(N-morpholino)propanesulfonic acid)), and different ratios of sucrose (Suc) to trehalose (Treh) to monosodium glutamate (MSG) to recombinant human serum albumin (rHSA).

[0018] [Figure 2A] Figures 2A and 2B show VCC multivariate data analysis (MVDA) in the accelerated stability study at the 1-month stability anchor point (Figure 2A) and the 6-month stability anchor point (Figure 2B). [Figure 2B] Figures 2A and 2B show VCC multivariate data analysis (MVDA) in the accelerated stability study at the 1-month stability anchor point (Figure 2A) and the 6-month stability anchor point (Figure 2B).

[0019] [Figure 3] Figure 3 shows VCC data for different OD levels and stabilizer combinations. The heading of each plot indicates the OD in the vial (2.0, 3.0, 10.0, 12.5, 15.0, 17.5, or 20.0), stabilizer (Buffer 2 (2.5% sucrose) or Buffer 5 (5% sucrose and amino acid mix)).

[0020] [Figure 4] Figure 4 shows VCC data for different OD levels and ratios of sucrose (Suc) to amino acid mix (AA mix) to recombinant human serum albumin (rHSA). The headings of each plot indicate the OD (2 or 10) in the vial, the ratio of sucrose (10, 5, 2.5):amino acid mix (present (1) or absent (0)):rHSA (0, 1, or 2.5).

[0021] [Figure 5] Figure 5 shows the residual moisture (RM) data for different OD levels and ratios of sucrose (Suc) to amino acid mix (AA mix) to recombinant human serum albumin (rHSA).

[0022] [Figure 6] FIG. 6 shows the residual moisture data for different percentages of sucrose (weight per volume) at different points in the freeze-drying cycle.

[0023] [Figure 7A]Figures 7A and 7B show post-lyophilization VCC data for Lm samples after the primary drying step, after ramping, or after the secondary drying step in the Lyo4 experiment, after storage at different temperatures for different amounts of time. Figure 7A shows the results for a formulation with 2.5% weight per volume (w / v) sucrose. Figure 7B shows the results for a formulation with 5% w / v sucrose. [Figure 7B] Figures 7A and 7B show post-lyophilization VCC data for Lm samples after the primary drying step, after ramping, or after the secondary drying step in the Lyo4 experiment, after storage at different temperatures for different amounts of time. Figure 7A shows the results for a formulation with 2.5% weight per volume (w / v) sucrose. Figure 7B shows the results for a formulation with 5% w / v sucrose.

[0024] [Figure 8] FIG. 8 shows post-lyophilization VCC data for Lm samples in the Lyo5 experiment that were stressed by a temperature shift in an ice bath before lyophilization, stressed by acid treatment to a lower pH before lyophilization, stressed by both a temperature shift and a pH shift before lyophilization, and Lm samples with no temperature or pH shift, after storage at different temperatures for different amounts of time.

[0025] [Figure 9] FIG. 9 shows the VCC data (percent of average pre-lyophilization VCC) before lyophilization (initial) and after lyophilization (all other samples) after storage at different temperatures for different amounts of time in the Lyo6 experiment.

[0026] [Figure 10] FIG. 10 shows the post-lyophilization VCC data (percent of mean pre-lyophilization VCC) before lyophilization and after storage at different temperatures for different amounts of time in the Lyo7 experiment.

[0027] [Figure 11]FIG. 11 shows post-lyophilization VCC data (percent of mean pre-lyophilization VCC) for fresh Lm samples (part A) and thawed frozen Lm samples (part B) after storage at different temperatures for different amounts of time in the Lyo8 experiment.

[0028] [Figure 12] FIG. 12 shows post-lyophilization VCC data after storage at different temperatures for different amounts of time in the Lyo9 experiment.

[0029] [Figure 13] FIG. 13 shows post-lyophilization VCC data for Lm samples stressed by a temperature shift in an ice bath before lyophilization (part B) and for Lm samples without a temperature shift (part A) after storage at different temperatures for different amounts of time in the Lyo10 experiment.

[0030] [Figure 14] FIG. 14 shows post-lyophilization VCC data after storage at different temperatures for different amounts of time in the Lyo11 experiment.

[0031] [Figure 15] Figure 15 shows post-lyophilization VCC data for fresh Lm samples (part A), frozen Lm samples thawed at 2-8 °C before lyophilization (part B), and frozen Lm samples thawed at 37 °C and incubated for 4 hours before lyophilization (part C) after storage at different temperatures for different amounts of time in the Lyo12 experiment.

[0032] [Figure 16] FIG. 16 shows post-lyophilization VCC data for fresh Lm samples (part A) and Lm samples stored at 2-8° C. for 3 days after storage at different temperatures for different amounts of time.

[0033] [Figure 17]FIG. 17 shows post-lyophilization VCC data after storage at different temperatures for different amounts of time for Lm samples under the following conditions: 2R vial, 1×10 9 VCCs, and 1.2 mL fill.

[0034] [Figure 18] FIG. 18 shows VCC data (CFU / mL) before lyophilization, after lyophilization, and at accelerated conditions at 30° C. for 1, 2, and 3 days (Tliq, Tlyo, T42h, T48h, and T72h, respectively).

[0035] [Figure 19] FIG. 19 shows the residual moisture content (RM) as a function of shelf temperature (SD temperature) in the secondary drying step.

[0036] [Figure 20] Figure 20 shows the drug substance manufacturing process flow for Axalimogene Filolisbac (ADXS-HPV).

[0037] [Figure 21] FIG. 21 shows a flow diagram for the manufacture of Axalimogene Filolisbac (ADXS-HPV) drug product.

[0038] [Figure 22A] FIG. 22A shows post-lyophilization VCC data (percent of mean pre-lyophilization VCC) before lyophilization and after storage at different temperatures for different amounts of time in the Lyo1 experiment.

[0039] [Figure 22B] Figure 22B shows the residual moisture immediately after freeze-drying and after 6 months at 2-8°C in the Lyo1 experiment.

[0040] [Figure 23A]FIG. 23A shows post-lyophilization VCC data (percent of mean pre-lyophilization VCC) before lyophilization and after storage at different temperatures for different amounts of time (months) in the Lyo2 experiment.

[0041] [Figure 23B] FIG. 23B shows the residual moisture immediately after freeze-drying and after 6 months at 2-8° C. in the Lyo2 experiment.

[0042] [Figure 24A] FIG. 24A shows the residual moisture (RM) after primary drying, ramping, and secondary drying using 2.5% sucrose after storage at different temperatures for different amounts of time in the Lyo4 experiment.

[0043] [Figure 24B] FIG. 24B shows the residual moisture (RM) after primary drying, ramping, and secondary drying using 5.0% sucrose after storage at different temperatures for different amounts of time in the Lyo4 experiment.

[0044] [Figure 25] FIG. 25 shows the residual moisture after various stress treatments after storage at different temperatures for different amounts of time in the Lyo5 experiment.

[0045] [Figure 26] FIG. 26 shows the residual moisture after temperature shift treatment before lyophilization and after storage at different temperatures for different amounts of time in the Lyo6 experiment.

[0046] [Figure 27] FIG. 27 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo7 experiment.

[0047] [Figure 28] FIG. 28 shows the residual moisture of samples that were immediately freeze-dried (part A) or frozen, thawed, and then freeze-dried (part B) after storage at different temperatures for different amounts of time in the Lyo8 experiment.

[0048] [Figure 29] FIG. 29 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo9 experiment.

[0049] [Figure 30] FIG. 30 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo10 experiment.

[0050] [Figure 31] FIG. 31 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo11 experiment.

[0051] [Figure 32] FIG. 32 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo12 experiment.

[0052] [Figure 33] FIG. 33 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo13 experiment.

[0053] [Figure 34] FIG. 34 shows the residual moisture after storage at different temperatures for different amounts of time in the Lyo14 experiment.

[0054] [Figure 35] FIG. 35 shows post-lyophilization VCC data as a percent of pre-lyophilization after storage at 30° C. for different amounts of time in a batch-scale experiment.

[0055] [Figure 36] FIG. 36 shows post-lyophilization VCC data as percent post-lyophilization after storage at 30° C. for different amounts of time in a batch-scale experiment.

[0056] [Figure 37]FIG. 37 shows the residual moisture (RM) vs. sample in the batch-scale experiment.

[0057] [Figure 38] Figure 38 shows the residual moisture (RM) after freeze-drying and storage at 30°C for 72 hours in a batch-scale experiment.

[0058] [Figure 39] FIG. 39 shows the bioactivity and INFγ induction of the lyophilized products compared to non-lyophilized bacteria and 10-mer controls in the Lyo11 experiment.

[0059] [Figure 40] 40A-40B show VCC data (A) and residual moisture (B) before freeze-drying, and immediately after freeze-drying and after freeze-drying after storage at 30° C. for 24, 48, or 72 hours in the WP3 experiment.

[0060] [Figure 41A] 41A-B show VCC data (A) and residual moisture (B) before freeze-drying, and immediately after freeze-drying and after freeze-drying after storage at 30° C. for 24, 48, or 72 hours in the WP6 experiment. [Figure 41B] 41A-B show VCC data (A) and residual moisture (B) before freeze-drying, and immediately after freeze-drying and after freeze-drying after storage at 30° C. for 24, 48, or 72 hours in the WP6 experiment.

[0061] [Figure 42] 42A-B show the lyophilized cake (A) and reconstitution time (B) immediately after lyophilization and after 24, 48, or 72 hours of storage at 30° C. in the WP7 experiment.

[0062] [Figure 43]FIG. 43 shows VCC data for the WP7 experiment before lyophilization, and immediately after lyophilization, and after lyophilization after storage at 30° C. for 24 hours, 72 hours, and 7 days.

[0063] [Figure 44A] 44A-B show VCC data (A) and percent viable cells (B) versus storage time at 30° C. for the WP7 experiment. [Figure 44B] 44A-B show VCC data (A) and percent viable cells (B) versus storage time at 30° C. for the WP7 experiment.

[0064] [Figure 45A] Figures 45A-B show CFU / mL (A) and percent viable cells (B) vs. time in the WP7 experiment. [Figure 45B] Figures 45A-B show CFU / mL (A) and percent viable cells (B) vs. time in the WP7 experiment.

[0065] [Figure 46] 46A-B show VCC data for the WP7 experiment before lyophilization, and immediately after lyophilization, and after lyophilization after storage at 30° C. for 24 and 72 hours.

[0066] [Figure 47] 47A-B show VCC data for the WP7 experiment before lyophilization, immediately after lyophilization, and after lyophilization after storage at 30° C. for 24 and 72 hours.

[0067] [Figure 48] Figure 48 shows a scatter plot of VCC and RM for the ADXS11-001 pilot batch.

[0068] [Figure 49]Figure 49 shows a scatter plot of VCC for lot #5329PD-17-01 (ADXS11-001 pilot batch).

[0069] [Figure 50] FIG. 50 shows a scatter plot of % live cells for lot #5329PD-17-01 (ADXS11-001 pilot batch).

[0070] [Figure 51] Figure 51 shows a scatter plot of pH for lot #5329PD-17-01 (ADXS11-001 pilot batch).

[0071] [Figure 52] Figure 52 shows a scatter plot of VCC for lot #5329PD-17-01 (ADXS11-001 pilot batch) stored at 30°C.

[0072] [Figure 53] Figure 53 shows a scatter plot of % viable cells for lot #5329PD-17-01 (ADXS11-001 pilot batch) stored at 30°C.

[0073] [Figure 54] FIG. 54 shows a chart illustrating the implantation and dosing schedule (ADXS11-001 pilot batch).

[0074] [Figure 55] FIG. 55 shows a graph illustrating that both lyophilized and clinical AXAL, at different doses, inhibit tumor growth in the TC-1 tumor model.

[0075] [Figure 56] FIG. 56 shows a graph illustrating that both lyophilized and clinical AXAL, at different doses, extend animal survival in the TC-1 tumor model.

[0076] [Figure 57] FIG. 57 shows a graph illustrating the reconstitution time of WP7 Cycle 3 compared to Cycle 1 and Cycle 2.

[0077] [Figure 58A] 58A-B show graphs illustrating MFI analysis of A0085 (A) and A1300 (B) in Tliq, Tlyo, and after storage at 30° C. for 24 and 72 hours. [Figure 58B] 58A-B show graphs illustrating MFI analysis of A0085 (A) and A1300 (B) in Tliq, Tlyo, and after storage at 30° C. for 24 and 72 hours.

[0078] [Figure 58C] Figures 58C-D show graphs illustrating MFI analysis of B0085 (C) and B1300 (D) in Tliq, Tlyo, and after storage at 30°C for 24 and 72 hours. [Figure 58D] Figures 58C-D show graphs illustrating MFI analysis of B0085 (C) and B1300 (D) in Tliq, Tlyo, and after storage at 30°C for 24 and 72 hours.

[0079] [Figure 59A] 59A-59B show graphs illustrating the RRM results for A.A0085 (200-fold dilution) and B.A1300 (5,000-fold dilution). [Figure 59B] 59A-59B show graphs illustrating the RRM results for A.A0085 (200-fold dilution) and B.A1300 (5,000-fold dilution).

[0080] [Figure 59C] Figures 59C-59D show graphs illustrating the RRM results for C.B0085 (200-fold dilution) and D.B1300 (5,000-fold dilution). [Figure 59D]Figures 59C-59D show graphs illustrating the RRM results for C.B0085 (200-fold dilution) and D.B1300 (5,000-fold dilution).

[0081] [Figure 60A] 60A-60B show graphs illustrating the distribution of negatively buoyant particles for A0085 (200-fold dilution) (A) and A1300 (5,000-fold dilution) (B). [Figure 60B] 60A-60B show graphs illustrating the distribution of negatively buoyant particles for A0085 (200-fold dilution) (A) and A1300 (5,000-fold dilution) (B).

[0082] [Figure 60C] Figures 60C-60D show graphs illustrating the distribution of negatively buoyant particles for B0085 (200-fold dilution) (C) and B1300 (5,000-fold dilution) (D). [Figure 60D] Figures 60C-60D show graphs illustrating the distribution of negatively buoyant particles for B0085 (200-fold dilution) (C) and B1300 (5,000-fold dilution) (D).

[0083] [Figure 61] Figure 61 shows a graph illustrating the Karl Fischer titration analysis of five vials of A0085, A1300, B0085 and B1300 Tlyo.

[0084] [Figure 62] Figure 62 shows a graph illustrating the results of Tliq, Tlyo, and VCC assays performed after 24, 48, and 72 hours of storage at 30°C. Prior vials were stored at -20°C for 7 days before analysis.

[0085] [Figure 63A] FIG. 63A shows a scatter plot illustrating VCC after WP7 cycle 3 in accelerated stability.

[0086] [Figure 63B] Figure 63B shows a scatter plot illustrating % viable cells after WP7 cycle 3 in accelerated stability.

[0087] [Figure 64] FIG. 64 shows a scatter plot illustrating the effect on VCC of repeated freeze / thaw cycles in BDS (1 L fill / 1 L LDPE bag) at various temperatures and VCC levels.

[0088] [Figure 65] FIG. 65 shows a scatter plot illustrating the effect on % viable cells of repeated freeze / thaw cycles in BDS (1 L fill / 1 L LDPE bag) at various temperatures and VCC levels. DETAILED DESCRIPTION OF THE INVENTION

[0089] definition The terms "protein," "polypeptide," and "peptide," used interchangeably herein, refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids, and amino acids that are chemically or biochemically modified or derivatized. These terms include modified polymers, such as polypeptides having modified peptide backbones.

[0090] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of a protein or polypeptide, which ends with an amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) which ends with a free carboxyl group (-COOH).

[0091] The term "fusion protein" refers to a protein comprising two or more peptides linked together by peptide or other chemical bonds. The peptides may also be directly linked to each other by peptide or other chemical bonds. For example, a chimeric molecule may be recombinantly expressed as a single-chain fusion protein. Alternatively, the peptides may be linked to each other by a "linker" between the two or more peptides, such as one or more amino acids or another suitable linker.

[0092] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, refer to a polymeric form of nucleotides of any length, containing ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0093] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react in a manner that unidirectionally attaches the 5' phosphate of one mononucleotide pentose ring to the 3' oxygen of its neighbor via a phosphodiester linkage to produce an oligonucleotide. An end of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. Nucleic acid sequences, even if internal to a larger oligonucleotide, are sometimes said to have 5' and 3' ends. In either a linear or circular DNA molecule, distinct elements are said to be "upstream" or 5' of the "downstream" or 3' element.

[0094] "Codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a more frequently or most frequently used codon in the host cell's genes while maintaining the native amino acid sequence for enhanced expression in a particular host cell. For example, a polynucleotide encoding a fusion polypeptide can be modified to replace a codon with a higher frequency of use compared to the nucleic acid sequence naturally occurring in a given Listeria cell or any other host cell. Codon usage tables are readily available, for example, from the "Codon Usage Database." The optimal codons utilized by L. monocytogenes for each amino acid are shown in US2007 / 0207170, which is incorporated herein by reference in its entirety for all purposes. These tables can be adapted in numerous ways. See Nakamura et al. (2000) Nucleic Acids Research, vol. 1, pp. 111-117, which is incorporated herein by reference in its entirety for all purposes. 28:292. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, eg, Gene Forge).

[0095] The term "plasmid" or "vector" includes any known delivery vector, including bacterial delivery vectors, viral delivery vectors, peptide immunotherapy delivery vectors, DNA immunotherapy delivery vectors, episomal plasmids, integrative plasmids, or phage vectors. The term "vector" refers to a construct that can deliver, and optionally express, one or more fusion polypeptides into a host cell.

[0096] The term "episomal plasmid" or "extrachromosomal plasmid" refers to a nucleic acid vector that is physically separate from chromosomal DNA (i.e., episomal or extrachromosomal and not integrated into the genome of the host cell) and replicates independently of chromosomal DNA. Plasmids can be linear or circular, single-stranded or double-stranded. Episomal plasmids can persist in multiple copies in the cytoplasm of the host cell (e.g., Listeria) if desired, resulting in amplification of any gene of interest within the episomal plasmid.

[0097] The term "genomically integrated" refers to a nucleic acid sequence that has been introduced into a cell and that, as a result of that introduction, is integrated into the genome of the cell and can be inherited by its progeny. Any protocol can be used for stable integration of a nucleic acid into the genome of a cell.

[0098] The term "stably maintained" refers to maintenance of a nucleic acid molecule or plasmid for at least 10 generations without detectable loss in the absence of selection (e.g., antibiotic selection). For example, this period can be at least 15, 20, 25, 30, 40, 50, 60, 80, 100, 150, 200, 300, or 500 generations. "Stable maintained" can refer to a nucleic acid molecule or plasmid that is stably maintained in cells in vitro (e.g., in culture), stably maintained in vivo, or both.

[0099] An "open reading frame" or "ORF" is a segment of DNA containing a sequence of bases that may potentially code for a protein. For example, an ORF can be located between the start-code sequence (initiation codon) and the stop codon sequence (termination codon) of a gene.

[0100] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a particular polynucleotide sequence. A promoter may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein modulate the transcription of an operably linked polynucleotide. The promoter may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a stage-specific promoter (e.g., a developmentally regulated promoter), or a region-specific promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO2013 / 176772, the entire contents of which are incorporated herein by reference.

[0101] "Operable linkage" or "operably linked" refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow for the potential for at least one of the components to mediate the function exerted by at least one of the other components. For example, a promoter can be operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences that are contiguous with each other or acting in trans (e.g., regulatory sequences can act at a distance to control transcription of a coding sequence).

[0102] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When sequence identity percentages are used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. Typically, this involves increasing the percentage sequence identity by scoring conservative substitutions as partial rather than full mismatches. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. Scoring of conservative substitutions is calculated, for example, according to the implementation of the program PC / GENE (Intelligenetics, Mountain View, California).

[0103] "Percentage sequence identity" refers to a value determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) over a comparison window, where a portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of matching positions, dividing this number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. Unless otherwise specified (for example, the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0104] Unless otherwise stated, sequence identity / similarity values ​​are calculated using the GAP algorithm with the following parameters: "Gap" refers to values ​​obtained using Version 10: % identity and % similarity for nucleotide sequences using a gap weight of 50, a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a gap weight of 8, a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program. "Equivalent program" includes any sequence comparison program that produces alignments with identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences at issue when compared to corresponding alignments produced by GAP Version 10.

[0105] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, or leucine, for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of a polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. In addition, the substitution of a basic residue, such as lysine, arginine, or histidine, for another, or the substitution of an acidic residue, such as aspartic acid or glutamic acid, for another, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, for a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, and / or the substitution of a polar residue for a non-polar residue. Typical amino acid classifications are summarized in Table 1 below.

[0106] [Table 1]

[0107] A "homologous" sequence (e.g., nucleic acid sequence) refers to a sequence that is identical or substantially similar to a known reference sequence, such that it is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence.

[0108] The term "wild-type" refers to an entity having a structure and / or activity as found in a normal state or situation (as opposed to a mutated, affected, altered, etc. state or situation). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0109] The term "isolated," with respect to proteins and nucleic acids, refers to proteins and nucleic acids that are relatively purified with respect to other bacterial, viral, or cellular components that may normally be present in situ, and extends to and includes substantially pure preparations of proteins and polynucleotides. The term "isolated" also includes proteins and nucleic acids that have no naturally occurring counterparts, that are chemically synthesized and therefore substantially uncontaminated by other proteins or nucleic acids, or that have been separated or purified from most other cellular components (e.g., other cellular proteins, polynucleotides, or cellular components) that naturally accompany them.

[0110] A "foreign" or "heterologous" molecule or sequence is a molecule or sequence that is not normally expressed by a cell or that is not normally present in that form within the cell. Normal presence includes presence with respect to a particular developmental stage and environmental conditions of the cell. For example, a foreign or heterologous molecule or sequence can include a mutant version of a corresponding endogenous sequence within the cell, or a sequence that corresponds to an endogenous sequence within the cell, but in a different form (i.e., not within the chromosome). A foreign or heterologous molecule or sequence within a particular cell can also include a molecule or sequence that originates from a species different from the reference species of the cell, or from a different organism within the same species. For example, in the case of a Listeria strain that expresses a heterologous polypeptide, the heterologous polypeptide can be a polypeptide that is not native or endogenous to the Listeria strain, i.e., a polypeptide not normally expressed by the Listeria strain, a polypeptide from a source other than the Listeria strain, or a polypeptide that originates from a different organism within the same species.

[0111] In contrast, an "endogenous" or "native" molecule or sequence is a molecule or sequence that is normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0112] The term "variant" refers to an amino acid or nucleic acid sequence (or organism or tissue) that differs from the majority of the population but is still similar enough to the common pattern to be considered one of them (e.g., a splice variant).

[0113] The term "isoform" refers to a version of a molecule (protein) that differs only slightly from another isoform or version (e.g., of the same protein). For example, protein isoforms can be produced from related but different genes, can arise from alternative splicing from the same gene, or can result from single nucleotide polymorphisms.

[0114] The term "fragment" when referring to a protein means a protein that is shorter than the full-length protein or that has fewer amino acids than the full-length protein. The term "fragment" when referring to a nucleic acid means a nucleic acid that is shorter than the full-length nucleic acid or that has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of the C-terminal portion of the protein), a C-terminal fragment (i.e., removal of the N-terminal portion of the protein), or an internal fragment. A fragment can also be, for example, a functional fragment or an immunogenic fragment.

[0115] The term "analog" when referring to a protein means a protein that differs from the naturally occurring protein by conservative amino acid differences, by modifications that do not affect amino acid sequence, or by both.

[0116] The term "functional" refers to the inherent ability of a protein or nucleic acid (or fragment, isoform, or variant thereof) to exhibit a biological activity or function. Such a biological activity or function may include, for example, the ability to elicit an immune response when administered to a subject. Such a biological activity or function may also include, for example, binding to an interaction partner. In the case of functional fragments, isoforms, or variants, these biological functions may, in fact, be altered (e.g., with respect to their specificity or selectivity) while retaining the basic biological function.

[0117] The term "immunogenicity" or "immunogenic" refers to the inherent ability of a molecule (e.g., a protein, nucleic acid, antigen, or organism) to induce an immune response in a subject when administered to the subject. Immunogenicity can be measured, for example, by a higher number of antibodies against the molecule, a higher density of antibodies against the molecule, a higher number of T cells specific to the molecule, a greater cytotoxic or helper T cell response against the molecule, etc.

[0118] The term "antigen" is used herein to refer to a substance that, when contacted with a subject or organism (e.g., when present in or detected by a subject or organism), results in a detectable immune response from the subject or organism. Antigens can be, for example, lipids, proteins, carbohydrates, nucleic acids, or combinations and variations thereof. For example, an "antigenic peptide" refers to a peptide that, when present in or detected by a subject or organism, elicits an increased immune response in the subject or organism. For example, such an "antigenic peptide" can include proteins that are loaded and presented on MHC class I and / or class II molecules on the surface of host cells and can be recognized or detected by the host's immune cells, resulting in an increased immune response against the protein. Such an immune response can also extend to other cells within the host, such as diseased cells (e.g., tumor or cancer cells) that express the same protein.

[0119] The term "epitope" refers to a site on an antigen that is recognized by the immune system (e.g., a site to which an antibody binds). Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. An epitope generally comprises at least three, more usually at least five or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 1, pp. 111-114, 1999, which is incorporated herein by reference in its entirety for all purposes. See Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0120] The term "mutation" refers to any change in the structure of a gene or protein. For example, mutations can occur as a result of deletion, insertion, substitution, or rearrangement of a chromosome or protein. An "insertion" changes the number of nucleotides in a gene or the number of amino acids in a protein by adding one or more additional nucleotides or amino acids. A "deletion" changes the number of nucleotides in a gene or the number of amino acids in a protein by reducing one or more additional nucleotides or amino acids.

[0121] A DNA "frameshift" mutation occurs when the addition or loss of a nucleotide changes the reading frame of a gene. A reading frame consists of a group of three bases, each of which codes for an amino acid. A frameshift mutation shifts the grouping of these bases, changing the code for the amino acid. The resulting protein is usually non-functional. Insertions and deletions are each frameshift mutations.

[0122] A "missense" mutation or substitution refers to a single amino acid change or single nucleotide point mutation in a protein that results in a change in the encoded amino acid. A single nucleotide point mutation that results in a single amino acid change is a "nonsynonymous" substitution in a DNA sequence. A nonsynonymous substitution can also result in a "nonsense" mutation that changes a codon to a premature stop codon, resulting in a truncated resulting protein. In contrast, a "synonymous" mutation in DNA is one that does not change the amino acid sequence of a protein (due to codon degeneracy).

[0123] The term "somatic mutation" includes genetic alterations acquired by cells other than germ cells (e.g., sperm or eggs). Such mutations are not heritable but can be passed on to the progeny of the mutated cell during cell division. In contrast, germ cell mutations occur in the germ line and can be passed on to future generations.

[0124] The term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment, such as a test tube.

[0125] The term "in vivo" refers to the natural environment (eg, a cell or organism or body) and to processes or reactions that occur within a natural environment.

[0126] The term "frozen-state glass transition temperature" (Tg') refers to the following: when heated, solutions of sugar glass undergo a second-order transition from a rigid state to a viscoelastic rubbery state. The temperature at which this glass-like transformation occurs is the glass transition temperature in the frozen state.

[0127] The term "solid state glass transition temperature" (Tg) refers to: Analogous to Tg', this is the temperature at which a freeze-dried glassy solid transforms into a viscoelastic rubbery state.

[0128] The term "collapse temperature" (Tc) refers to the maximum temperature that a product can withstand during primary drying without losing its physical structure.

[0129] The term "drug substance" (DS) refers to an active ingredient. A drug substance refers to any component of a drug product intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human or other animal body. Active ingredients include components of a product that may be chemically altered during the manufacturing of the drug product and may be present in the drug product in a modified form intended to provide the specified activity or effect. For example, Lm (e.g., ADXS-HPV or ADXS-HER2) is considered a drug substance.

[0130] The term "bulk drug substance" (BDS) refers to any substance that is intended for use in a drug and that, when used in the manufacture, processing, or packaging of a drug, becomes the active ingredient or finished dosage form of the drug, but the term does not include intermediates used in the synthesis of such a substance.

[0131] The term "drug product" (DP) refers to a finished dosage form, e.g., tablet, capsule, or solution, containing an active pharmaceutical ingredient, typically, but not necessarily, in association with an active ingredient. For example, lyophilized Lm (e.g., ADXS-HPV or ADXS-HER2) is considered a drug product.

[0132] A composition or method that "comprising" or "including" one or more listed elements may also include other elements not specifically listed. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other components.

[0133] Designations of ranges of values ​​include all integers within or defining the range, and all sub-ranges defined by integers within the range.

[0134] Unless otherwise clear from the context, the term "about" encompasses values ​​within the stated value or standard error of measurement (e.g., SEM) of ±0.5%, 1%, 5%, or 10% variation from the stated value.

[0135] The singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an antigen" or "at least one antigen" can include multiple antigens, including mixtures thereof.

[0136] Statistically significant means p≦0.05. Detailed Description I. Summary

[0137] Disclosed herein are compositions and methods relating to stable lyophilized pharmaceutical formulations prepared by lyophilizing an aqueous formulation containing bacteria or Listeria strains, such as Listeria monocytogenes. In some embodiments, the lyophilized formulations are stable at 4° C. or −20° C. for at least 6 months, at least 1 year, or at least 2 years. In some embodiments, the lyophilized formulations are suitable for parenteral administration, such as intravenous injection.

[0138] Frozen liquid formulations currently used for therapeutics, including L. monocytogenes, are stored and shipped at -80°C. This low temperature presents supply chain challenges, particularly in South American and African countries, for both shipping materials and storing them at clinical sites. Therefore, having a refrigerated or -20°C supply chain is desirable. Optimizing the manufacturing process described herein can produce stable drug products that can be maintained at higher temperatures. Counterintuitively, the examples described herein demonstrate that higher residual moisture (e.g., higher than typical target residual moisture levels, e.g., about 2.5%, 3.0%, or 3.5% in one embodiment) improved the stability of the lyophilized product. Similarly, counterintuitively, higher shelf temperatures during the primary drying step (e.g., well above Tg, e.g., about -17°C to about -19°C or about -18°C in one embodiment) improved the stability of the lyophilized product. Additionally, preconditioning the cells by heat shock prior to lyophilization improved the stability of the lyophilized product. Additionally, the use of higher viable bacterial concentrations (viable cell count, or VCC) results in improved stability of lyophilized drug products compared to lower VCC. Additionally, counterintuitively, thawing frozen drug substance at approximately 37°C prior to lyophilization improved the stability of lyophilized drug products compared to thawing at room temperature or 2-8°C. These process improvements improve stability at higher temperatures compared to liquid frozen formulations, thereby enabling a higher temperature supply chain. This allows for a more manageable supply chain and distribution to countries where storage at -80°C is not feasible.

[0139] In addition to optimizing the residual moisture in the lyophilized cake (e.g., using a higher than normal target residual moisture level, e.g., about 3.5% in one embodiment, which can be achieved by varying the secondary drying temperature and, if necessary, the secondary drying time), the process of inducing a stress response in L. monocytogenes cells by temperature shift prior to lyophilization improves the stability at higher temperatures for the lyophilized drug product. Similarly, using a phosphate buffer and a lower than normal level of sucrose (e.g., about 2.5% in one embodiment) can improve the stability of the lyophilized drug product at higher temperatures. The use of a formulation comprising a lyophilized drug product containing sucrose (w / v) and a high primary drying step temperature (e.g., about -18°C in one embodiment) improves the stability of the lyophilized drug product at higher temperatures, including -20°C, 2-8°C, and even room temperature (about 20°C to about 25°C, or about 20°C, about 23°C, or about 25°C). These process improvements improve stability at higher temperatures compared to liquid frozen formulations, thereby enabling a higher temperature supply chain. This allows for a more manageable supply chain and distribution to countries where storage at -80°C is not feasible. II. Freeze-drying of bacteria or Listeria

[0140] Freeze-drying can be divided into three steps: freezing, primary drying, and secondary drying. Water is frozen in the first step, so that dissolved components of the formulation remain in a residual liquid (freeze concentrate). At the point of maximum ice formation, the freeze concentrate solidifies between the ice crystals that make up the lattice. Under appropriate freeze-drying conditions, the ice is removed by sublimation during primary drying, leaving behind a residual freeze concentrate with the same physical and chemical structure as when the ice was present. Residual water in the freeze concentrate is removed in the secondary drying step.

[0141] Lyophilization involves manipulating the temperature and pressure of a solution to transition the solvent phase directly from a frozen state to a gaseous state without passing through a liquid / liquid phase. This is achieved by cooling the solution below the triple point of water and reducing the pressure. This allows for the removal of the solvent from the product without exposing it to intense heat. During the freezing stage, the formulation is cooled. Pure crystalline ice forms from the liquid, causing the remainder of the liquid to freeze-concentrate to a more viscous state, inhibiting further crystallization. Ultimately, this highly concentrated, viscous solution solidifies, resulting in an amorphous, crystalline, or hybrid amorphous-crystalline phase. During the primary drying stage, the ice formed during freezing is removed by sublimation under vacuum at temperatures below ambient temperature. Throughout this stage, the product is maintained in a solid state below its collapse temperature, which is the glass transition temperature (Tg') for amorphous products or the eutectic temperature (Te) for crystalline products, in order to dry the product while retaining the structure established during the freezing step. During the secondary drying stage, the relatively small amount of bound water remaining in the matrix is ​​removed by desorption. During this stage, shelf and product temperatures are elevated to promote a reasonable desorption rate and to achieve the desired residual moisture.

[0142] The target profile for lyophilized drug products is one that produces a well-defined cake with a target residual moisture content that is stable at either 2-8°C or -20°C and retains the same potency and bioactivity as the liquid frozen formulation. Protective strategies that can improve bacterial viability during freeze-drying include, for example, adding excipients to the drying medium, controlling process parameters, prestressing bacterial samples before freeze-drying, and altering bacterial fermentation conditions. However, the efficiency of these strategies is strain-dependent because different strains have different intrinsic tolerances to the drying process. Even among closely related bacterial strains, some may tolerate the freeze-drying process much better than others. This strain dependency makes it difficult to draw general conclusions or guidance.

[0143] Methods for producing a lyophilized composition comprising bacteria or Listeria strains are provided herein. Such methods can include providing a composition comprising bacteria or Listeria strains in a formulation comprising a buffer, cooling the composition in a freezing step, exposing the cooled composition to a vacuum and a first elevated temperature in a primary drying step, and exposing the composition from the primary drying step to a vacuum and a second elevated temperature in a secondary drying step, thereby producing the lyophilized composition.

[0144] In some such methods, the bacteria or Listeria strains used in the composition are frozen Listeria strains that are thawed prior to the freezing step. Examples of such preconditioning steps are described in more detail elsewhere herein. In particular examples, the frozen bacteria or Listeria strains can be thawed at a temperature of about 2°C to about 37°C, about 20°C to about 37°C, about 23°C to about 37°C, about 25°C to about 37°C, about 32°C to about 37°C, or about 37°C. Optionally, thawing is for about 8 hours or less. Optionally, the thawed bacteria or Listeria strains are held at a temperature of about 2°C to about 8°C for about 24 hours or less. In particular examples, the concentration of the thawed bacteria or Listeria strains is about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 It can be between colony forming units (CFU).

[0145] In some such methods, the formulation comprises a buffer and sucrose. For example, the formulation buffer may comprise about 1% to about 5% w / v sucrose, about 2% to about 3% w / v sucrose, or about 2.5% w / v sucrose. Optionally, the formulation does not contain other excipients such as trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA).

[0146] In some embodiments, the formulation contains about 1 x 10 per milliliter 9 ~Approx. 1×1010 Contains colony forming units (CFU) of bacteria or Listeria.

[0147] In some such methods, the holding temperature in the primary drying step is between about -10°C and about -30°C, between about -12°C and about -22°C, between about -17°C and about -19°C, or about -18°C.

[0148] In some such methods, the residual moisture in the lyophilized composition is at least about 2.5%, at least about 3%, or at least about 3.5%. In some such methods, the residual moisture is between about 1% and about 5%, or between about 2% and about 4%.

[0149] In some such methods, the lyophilized composition exhibits a viability of at least about 60%, 70%, 80%, or 90% after storage between about -20°C and about 4°C, or after storage at about -20°C or about 4°C for about 6 months, 12 months, 18 months, or 24 months.

[0150] Some such methods include the steps of: (a) providing a composition comprising a bacterium or Listeria strain in a formulation comprising a buffer and sucrose; (b) cooling the composition provided in step (a) at a holding temperature of between about −32° C. and about −80° C. in a freezing step; (c) exposing the composition produced by step (b) to a vacuum at a holding temperature of between about −10° C. and about −30° C. in a primary drying step; and (d) exposing the composition produced by step (c) to a vacuum at a holding temperature of between about −5° C. and about 25° C. in a secondary drying step.

[0151] Further embodiments for cell preconditioning, formulation, freezing steps, primary drying steps, secondary drying steps, and lyophilized products are provided below. A. Preconditioning of bacteria or Listeria

[0152] The culture of bacteria or Listeria strains used in the freeze-drying methods disclosed herein can be from frozen stocks, from starter cultures, or from colonies (e.g., freshly cultured bacteria or Listeria).

[0153] Provided herein are methods for preparing frozen bacteria or Listeria strains for lyophilization, the methods including thawing the frozen bacteria or Listeria strains. If the bacteria or Listeria strains are from a frozen stock, they can be thawed by any means. The temperature and time for thawing can affect stability. Identifying appropriate conditions for thawing a frozen drug substance allows for freezing and preserving the drug substance before lyophilization. Ensuring high-quality, healthy cells resulting from thawing ensures that the resulting lyophilized drug product is also of sufficient quality. In one example, it can be thawed at about -4°C, about 2-8°C, or about 4°C and incubated for, for example, about 0.5, 1, 2, 3, 4 hours, or longer. In another example, it can be thawed at about 37°C and incubated for, for example, about 0.5, 1, 2, 3, 4 hours, or longer.

[0154] In one example, frozen bacteria or Listeria strains can be thawed at a temperature between about 4°C and about 37°C, between about 10°C and about 37°C, between about 15°C and about 37°C, between about 20°C and about 37°C, between about 23°C and about 37°C, between about 25°C and about 37°C, between about 25°C and about 37°C, between about 30°C and about 37°C, between about 32°C and about 37°C, between about 32°C and about 42°C, between about 34°C and about 40°C, between about 35°C and about 39°C, between about 36°C and 38°C, or about 37°C.

[0155] Frozen bacteria or Listeria strains can be thawed for, e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours, between about 0.5 and about 8 hours, between about 1 and about 8 hours, between about 2 and about 8 hours, between about 3 and about 8 hours, between about 4 and about 8 hours, between about 5 and about 8 hours, between about 6 and about 8 hours, or between about 7 and about 8 hours. Alternatively, frozen bacteria or Listeria strains can be thawed for, e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours or less.

[0156] The frozen bacteria or Listeria strains that are thawed may be in, or may be thawed in, a bacteria or Listeria lyophilized formulation, such as those disclosed in more detail elsewhere herein.

[0157] Frozen bacteria or Listeria strains can be maintained at a certain temperature after thawing. For example, frozen bacteria or Listeria strains can be maintained at a temperature between about 2°C and about 8°C after thawing. Frozen bacteria or Listeria strains can be maintained for, for example, about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or for about 0.5 to about 24 hours, about 1 to about 24 hours, about 2 to about 24 hours, about 5 to about 24 hours, about 10 to about 24 hours, or about 12 to about 24 hours. Alternatively, frozen bacteria or Listeria strains can be held for, e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or less. In a particular example, frozen bacteria or Listeria strains are thawed at a temperature of about 37°C for about 8 hours or less and held at a temperature between about 2°C and about 8°C for about 24 hours or less.

[0158] The concentration of the thawed bacteria or Listeria strain can be any suitable concentration. For example, the concentration can be about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 It can be between colony forming units (CFU).

[0159] The culture used for lyophilization can be in any growth phase, for example, the culture can be in mid-log phase, near mid-log phase, or another growth phase.

[0160] The nutrient medium utilized to grow the culture of bacteria or Listeria strains can be any suitable nutrient medium. Examples of suitable media include, for example, Luria Broth (LB: Luria-Bertani Broth); Terrific Broth (TB); animal-free modified terrific broth; or defined media. The bacteria or Listeria strains can be cultured by any known means for growing cells. For example, the growth step can be performed using shake flasks (e.g., baffled shake flasks), batch fermentors, stirred tanks or flasks, airlift fermentors, fed-batch, continuous cell reactors, immobilized cell reactors, or any other means for growing bacteria.

[0161] If desired, a constant pH is maintained during growth of the culture (e.g., in a batch fermentor). For example, the pH can be maintained at about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. Similarly, the pH can be, for example, about 6.5 to about 7.5, about 6.0 to about 8.0, about 6.0 to about 7.0, about 6.0 to about 7.0, or about 6.5 to about 7.5. Alternatively, immediately after harvesting the cells from the bioreactor, the pH can be lowered by the addition of acid to induce a stress response that can activate a set of genes that can better prepare the cells for lyophilization.

[0162] If desired, a constant temperature can be maintained during growth of the culture. For example, the temperature can be maintained at about 37°C. Alternatively, the temperature can be maintained at about 25°C, about 27°C, about 28°C, about 30°C, about 32°C, about 34°C, about 35°C, about 36°C, about 38°C, or about 39°C. Alternatively, immediately after harvesting the cells from the bioreactor, the temperature can be lowered by placing the cells in an ice bath (e.g., about 0°C or about 4°C) to induce a stress response that can activate a set of genes that can better prepare the cells for freeze-drying.

[0163] If desired, a constant dissolved oxygen concentration can be maintained during growth of the culture. For example, the dissolved oxygen concentration can be maintained at 20% saturation, 15% saturation, 16% saturation, 18% saturation, 22% saturation, 25% saturation, 30% saturation, 35% saturation, 40% saturation, 45% saturation, 50% saturation, 55% saturation, 60% saturation, 65% saturation, 70% saturation, 75% saturation, 80% saturation, 85% saturation, 90% saturation, 95% saturation, 100% saturation, or near 100% saturation.

[0164] Bacteria or Listeria strains can be passaged through an animal host, if necessary, before lyophilization. Such passage can maximize the effectiveness of the Listeria strain as a vaccine vector, stabilize the immunogenicity of the Listeria strain, stabilize the virulence of the Listeria strain, increase the immunogenicity of the Listeria strain, increase the virulence of the Listeria strain, eliminate unstable substrains of the Listeria strain, or prevent the spread of unstable substrains of the Listeria strain. Methods for passage of Listeria strains through an animal host are well known and are described, for example, in US2006 / 0233835, the entire contents of which are incorporated herein by reference for all purposes. B. Bacteria or Listeria freeze-dried preparation

[0165] Prior to lyophilization, the bacteria or Listeria strains can be provided in a suspension (formulation) containing a buffer and excipients. The design of the lyophilized formulation can depend on the requirements of the active pharmaceutical ingredient and the intended route of administration. The formulation can consist of a buffer and one or more excipients that perform one or more functions. Such excipients can be, for example, pH adjusters, bulking agents (e.g., sucrose, mannitol, maltose, trehalose, dextrose, and lactose), stabilizers, such as cryoprotectants (e.g., PEG) and lyoprotectants (e.g., disaccharides), or tonicity adjusters (e.g., NaCl, mannitol, sucrose, glycine, and glycerol).

[0166] The buffer can be any suitable buffer. The buffer can stabilize the pH of the formulation. For example, the buffer can be a phosphate buffer, a Tris buffer, a histidine buffer, a citrate buffer, or a MOPS (3-(N-morpholino)propanesulfonic acid) buffer. In a particular example, the buffer is a phosphate buffer. Phosphate buffers are often avoided in the development of lyophilized formulations because phosphate buffers, such as sodium phosphate, can cause extreme pH changes during freezing. For this reason, low-concentration buffers that cause little pH change during freezing, such as Tris, citrate, and histidine buffers, are often used. However, as shown elsewhere herein, suitable survival levels of Listeria monocytogenes are achieved using phosphate buffers. Some such buffers have a KH2PO4 (anhydrous) concentration of about 0.1-0.3 g / L, 0.12-0.28 g / L, 0.14-0.26 g / L, 0.16-0.24 g / L, 0.18-0.22 g / L, 0.19-0.21 g / L, or 0.2 g / L. Some such buffers have a Na2HPO4 (anhydrous) concentration of about 1.0-1.3 g / L, 1.02-1.28 g / L, 1.04-1.26 g / L, 1.06-1.24 g / L, 1.08-1.22 g / L, 1.1-1.2 g / L, 1.12-1.18 g / L, 1.14-1.16 g / L, or 1.15 g / L. Some such buffers have a pH of about 5-20, 6-18, 7-16, 8-14, 9-12, 9-11, or 10 mM. Some such buffers have a pH of about 6.8-7.6, 6.9-7.5, 7.0-7.4, 7.1-7.3, or 7.2. As an example, a phosphate buffer can have between about 0.19-0.21 g / L (e.g., 0.2 g / L) of KH2PO4 (anhydrous), between about 1.14-1.16 g / L (e.g., 1.15 g / L) of Na2HPO4 (anhydrous), and a pH of about 7.1-7.3 (e.g., 7.2).

[0167] Excipients such as cryoprotectants and lyoprotectants can be added to the formulation to protect bacteria or Listeria strains during the freeze-drying process. Cryoprotectants are water-soluble chemicals that lower the melting point of water. When ice crystals form, bacterial cells are compressed into the unfrozen fraction. The addition of a cryoprotectant can expand the unfrozen section, providing bacterial cells with more space, which can reduce cell damage due to mechanical or osmotic stress. Lyoprotectants can protect bacterial cells when water is removed during the drying step. Some sugars, such as sucrose and trehalose, can act as both cryoprotectants and lyoprotectants. The use of nonfat milk can also provide a protective effect. Other examples of excipients include glucose, maltose, lactose, mannitol, glycine, glycerol, sodium chloride, yeast extract, dextran, dextrose, polydextrose, monosodium glutamate, maltodextrin, antioxidants (e.g., ascorbic acid), saccharides, disaccharides, sugars, etc. In one example, the excipients used in the formulation include various combinations of sucrose, trehalose, monosodium glutamate (MSG), recombinant human serum albumin (rHSA), and amino acid mix. In particular examples, the excipients comprise, consist essentially of, or consist of sucrose, such as about 5% w / v (weight per unit volume) sucrose or about 2.5% w / v sucrose. For example, the formulation buffer may contain about 1% to about 5% w / v sucrose, about 2% to about 3% w / v sucrose, or about 2.5% w / v sucrose.

[0168] Optionally, the excipient does not include one or more or all of: trehalose, MSG, rHSA, amino acid mix, nonfat milk, glucose, maltose, lactose, mannitol, glycine, glycerol, sodium chloride, yeast extract, dextran, dextrose, polydextrose, monosodium glutamate, maltodextrin, ascorbic acid, sugars other than sucrose, disaccharides other than sucrose, sugars other than sucrose, or antioxidants. Optionally, the excipient does not include one or more or all of: trehalose, MSG, and rHSA.

[0169] The concentration of bacteria or Listeria in the formulation can be any suitable concentration. For example, the concentration can be about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 It can be between colony forming units (CFU). C. Freezing step

[0170] The first step in freeze-drying is the freezing step. During this step, the formulation is cooled. This can be achieved, for example, by lowering the shelf temperature of the freeze dryer (i.e., lowering the shelf temperature) in a shelf-type freeze dryer. Ice crystals form during freezing, which can damage bacteria. Ice crystal growth depends on the freezing rate and temperature. In some embodiments, a faster freezing rate is utilized. A faster freezing rate results in the formation of smaller ice crystals compared to a slower freezing rate, thus reducing cell damage. Ice crystal formation can be harmful to bacteria. When water crystallizes, solutes in the remaining unfrozen fraction concentrate, which can lead to chemical and osmotic damage. Freezing bacteria at a lower temperature corresponds to a faster freezing rate and results in smaller ice crystals, which should limit cell damage, but a faster freezing rate does not necessarily correspond to the best viability results. Optimal freezing conditions may vary depending on the protectant used in the formulation and the bacterial strain.

[0171] The holding temperature (e.g., shelf temperature) for the freezing step can be reached by lowering the temperature (e.g., shelf temperature) at a rate of, for example, about 0.2°C to about 2.0°C per minute. Alternatively, the holding temperature (e.g., shelf temperature) for the freezing step can be reached by lowering the temperature (e.g., shelf temperature) at a rate of, for example, about 0.2°C to about 1.8°C per minute, about 0.4°C to about 1.6°C per minute, about 0.6°C to about 1.4°C per minute, about 0.8°C to about 1.2°C per minute, or about 0.9°C to about 1.1°C per minute. For example, the temperature can be reduced to the freezing temperature at a rate of about 0.2°C, about 0.3°C, about 0.4°C, about 0.5°C, about 0.6°C, about 0.7°C, about 0.8°C, about 0.9°C, about 1.0°C, about 1.1°C, about 1.2°C, about 1.3°C, about 1.4°C, about 1.5°C, about 1.6°C, about 1.7°C, about 1.8°C, about 1.9°C, or about 2.0°C per minute. In a particular example, the hold temperature of the freezing step is reached by reducing the temperature to the hold temperature at a rate of about 1°C per minute.

[0172] The freezing step can be any suitable time step for freezing bacteria or Listeria strains. Similarly, the temperature can be held at the freezing temperature for any suitable time for freezing bacteria or Listeria strains. For example, the freezing step can be about 2 to about 6, about 2.5 to about 6, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1.5 to about 2.5, about 1.5 to about 5.5, about 2 to about 5, about 2.5 to about 4.5, about 3 to about 4, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, or about 6 hours, or the temperature can be held at the freezing temperature for the aforementioned times. In a particular example, the freezing step can be a 3.5 hour step, or the temperature can be held at the freezing temperature for 3.5 hours. In another specific example, the freezing step can be a 2 hour step, or the temperature can be held at the freezing temperature for 2 hours. In another specific example, the freezing step can be a 1.5 hour step, or the temperature can be held at the freezing temperature for 1.5 hours. In another specific example, the total freezing step (e.g., raising the temperature to the freezing temperature and then holding at the holding temperature) lasts for about 3.5 to 4.5 hours, or about 3.5 hours.

[0173] The freezing temperature (i.e., holding temperature) can be any temperature suitable for freezing bacteria or Listeria strains. In some embodiments, the freezing temperature (e.g., shelf temperature) is such that the temperature of the formulation is below the glass transition temperature of the solution, which for a sucrose formulation may be, for example, about -32°C. Temperatures higher than this temperature may not truly freeze the solution, in which case the solution may undergo collapse during lyophilization, potentially leading to reduced viability. For example, the temperature (e.g., shelf temperature) can be between about −49° C. and about −25° C., about −47° C. and about −40° C., about −45° C. and about −35° C., about −10° C. and about −80° C., about −15° C. and about −75° C., about −20° C. and about −70° C., about −25° C. and about −65° C., about −30° C. and about −60° C., about −35° C. and about −55° C., about −40° C. and about −50° C., about −41° C. and about −49° C., about −42° C. and about −48° C., about −43° C. and about −47° C., or about −44° C. and about −46° C. In a particular example, the freezing temperature can be about −45° C. In another example, the temperature is between about -49°C and about -32°C, about -47°C and about -40°C, about -45°C and about -35°C, about -32°C and about -80°C, about -32°C and about -75°C, about -32°C and about -70°C, about -32°C and about -65°C, about -32°C and about -60°C, -49°C and about -33°C, about -33°C and about -80°C, about -32°C and about -75°C, about -32°C and about -70°C, about -32°C and about -65°C, about -32°C and about -60°C, about -49°C and about -33°C, about -33°C and about -80°C, about -32°C and about -75°C, about -32°C and about -7 The freezing temperature may be about -39°C, about -33°C to about -75°C, about -33°C to about -70°C, about -33°C to about -65°C, about -33°C to about -60°C, about -35°C to about -55°C, about -40°C to about -50°C, about -41°C to about -49°C, about -42°C to about -48°C, about -43°C to about -47°C, or about -44°C to about -46°C. In one example, the freezing temperature may be about -39°C. In another example, the freezing temperature may be about -45°C. In a specific example, the holding temperature in the freezing step is between about -40°C to about -50°C (e.g., about -45°C), the freezing step includes lowering the temperature to the holding temperature at a rate of about 1°C per minute, and the cooling in the freezing step is for about 2 hours to about 4 hours (e.g., the freezing step includes holding the composition at the holding temperature for about 2 hours). D. Primary Drying Step

[0174] The second step in freeze-drying is the primary drying step, in which the composition containing the bacteria or Listeria strains produced by the freezing step is exposed to a vacuum at an elevated temperature, during which the frozen water is removed by sublimation under vacuum.

[0175] For example, the temperature for the primary drying step can be reached by increasing the temperature (e.g., shelf temperature) at a rate of about 0.2°C to about 2.0°C per minute. Alternatively, the holding temperature for the primary drying step (e.g., shelf temperature) can be reached by increasing the temperature (e.g., shelf temperature) at a rate of about 0.2°C to about 1.8°C per minute, about 0.4°C to about 1.6°C per minute, about 0.6°C to about 1.4°C per minute, about 0.8°C to about 1.2°C per minute, or about 0.9°C to about 1.1°C per minute. For example, the temperature can be increased to the primary drying temperature at a rate of about 0.2°C, about 0.3°C, about 0.4°C, about 0.5°C, about 0.6°C, about 0.7°C, about 0.8°C, about 0.9°C, about 1.0°C, about 1.1°C, about 1.2°C, about 1.3°C, about 1.4°C, about 1.5°C, about 1.6°C, about 1.7°C, about 1.8°C, about 1.9°C, or about 2.0°C per minute. In a particular example, the hold temperature of the primary drying step is reached by increasing the temperature to the hold temperature at a rate of about 1°C per minute.

[0176] The primary drying step can be any suitable time step. Similarly, the holding temperature (e.g., shelf temperature) can be held at the primary drying temperature for any suitable time. The temperature should be held at the primary drying temperature until primary drying is complete. This time can vary depending on the freeze dryer, vial size, fill volume, number of vials, pressure, and other variables. For example, the end of primary drying can be determined when the product temperature rises to or above the shelf temperature. This can also be determined by a pressure rise test, for example, in which the freeze-drying chamber is isolated from the vacuum pump and the amount of pressure rise due to continued water sublimation is determined. For example, the primary drying step can be about 10 to about 29, about 29 to about 42, about 36, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, or about 20 to about 30 hours, or the temperature can be held at the primary drying temperature for the aforementioned times. In particular examples, the primary drying step can be about a 25 to about 35, about 26 to about 34, about 27 to about 33, about 28 to about 32, about 29 to about 31, or about 30 hour step, or the temperature can be held at the primary drying temperature for the aforementioned times. In another particular example, the primary drying step can be about a 20 to about 30, about 21 to about 30, about 22 to about 30, about 23 to about 29, about 24 to about 28, about 25 to about 27, or about 26 hour step, or the temperature can be held at the primary drying temperature for the aforementioned times.

[0177] The primary drying step begins when a probe in the freeze dryer (e.g., a probe for a cold spot in the freeze dryer, such as one in the center of the freeze dryer) reaches a primary drying hold temperature or T s The drying step can be a step defined as about 8 to about 20, about 9 to about 19, about 10 to about 18, about 11 to about 17, about 12 to about 16, about 13 to about 15, or about 14 hours after passing a set point (e.g., about -18°C), or the temperature can be held at the primary drying hold temperature for the aforementioned period. Alternatively, the drying step can be a step defined as a step in which the composition to be lyophilized (e.g., a sample of the composition in the cold spot of the lyophilizer, or all samples of the composition in the lyophilizer) is heated to the primary drying hold temperature or T sThe primary drying step can be defined as a time period of about 8 to about 20, about 9 to about 19, about 10 to about 18, about 11 to about 17, about 12 to about 16, about 13 to about 15, or about 14 hours after reaching a set point (e.g., about -18°C), or the temperature can be held at the primary drying hold temperature for the aforementioned period. In certain examples, the primary drying step can be, for example, about 30 hours, when a probe in the freeze dryer (e.g., a probe for a cold spot in the freeze dryer, such as one in the center of the freeze dryer) reaches the primary drying hold temperature or T s After passing a set point (e.g., about −18° C.) or the composition being lyophilized (e.g., a sample of the composition in the cold spot of the lyophilizer, or all samples of the composition in the lyophilizer) reaches the primary drying hold temperature or T s This may be a step with a period defined as about 14 hours after reaching the set point (eg, about -18°C), or the temperature may be held at the primary dry hold temperature for the aforementioned period.

[0178] The end of the primary drying step is determined when a probe in the freeze dryer (e.g., a probe in a cold spot in the freeze dryer, such as in the center of the freeze dryer) reaches the primary drying hold temperature or T s The primary drying step can be completed about 8 to about 20, about 9 to about 19, about 10 to about 18, about 11 to about 17, about 12 to about 16, about 13 to about 15, or about 14 hours after passing the set point (e.g., about -18°C). Alternatively, the primary drying step can be completed when the composition to be lyophilized (e.g., a sample of the composition in the cold spot of the lyophilizer, or all samples of the composition in the lyophilizer) reaches the primary drying hold temperature or T s The end of the primary drying step can be about 8 to about 20, about 9 to about 19, about 10 to about 18, about 11 to about 17, about 12 to about 16, about 13 to about 15, or about 14 hours after the set point (e.g., about -18°C) is reached. In certain examples, the end of the primary drying step can be, for example, about 30 hours, when a probe in the freeze dryer (e.g., a probe for a cold spot in the freeze dryer, such as one in the center of the freeze dryer) reaches the primary drying hold temperature or T sAbout 14 hours after passing the set point (e.g., about −18° C.), or when the composition being lyophilized (e.g., a sample of the composition in the cold spot of the lyophilizer, or all samples of the composition in the lyophilizer) reaches the primary drying hold temperature or T s This can be about 14 hours after the set point (eg, about -18°C) is reached.

[0179] The primary drying temperature (i.e., shelf temperature or hold temperature) can be any suitable temperature for drying bacteria or Listeria strains. For example, the hold temperature can be between about 0°C and about -30°C, 0°C to about -19°C, about -5°C to about -30°C, about -10°C to about -25°C, about -15°C to about -20°C, about -17°C to about -19°C, about -12°C to about -30°C, about -12°C to about -24°C, about -12°C to about -22°C, about -14°C to about -22°C, about -15°C to about -21°C, about -16°C to about -20°C, about -17°C to about -19°C, about -18°C to about -22°C, about -30 ... The angle may be about -29°, about -28°, about -27°, about -26°, about -25°, about -24°, about -23°, about -22°, about -21°, about -20°, about -19°, about -18°, about -17°, about -16°, about -15°, about -14°, about -13°, about -12°, about -11°, about -10°, about -9°, about -8°, about -7°, about -6°, about -5°, about -4°, about -3°, about -2°, about -1°, or about 0°. For example, the temperature can be about −30° C., about −29° C., about −28° C., about −27° C., about −26° C., about −25° C., about −24° C., about −23° C., about −22° C., about −21° C., about −20° C., about −19° C., about −18° C., about −17° C., about −16° C., about −15° C., about −14° C., about −13° C., about −12° C., about −11° C., or about −10° C. or less. In certain examples, the primary drying temperature can be about −25° C. to about −35° C., about −26° C. to about −37° C., about −27° C. to about −33° C., about −28° C. to about −32° C., about −29° C. to about −31° C., or about −30° C. In a specific example, the primary drying temperature can be about −17° C. to about −27° C., about −18° C. to about −26° C., about −19° C. to about −25° C., about −20° C. to about −24° C., about −21° C. to about −23° C., or about −22° C. In another specific example, the primary drying temperature can be about −7° C. to about −17° C., about −8° C. to about −16° C., about −9° C. to about −15° C., about −10° C. to about −14° C., about −11° C. to about −13° C., or about −12° C. In another specific example, the primary drying temperature can be about −13° C. to about −23° C., about −14° C. to about −22° C., about −15° C. to about −21° C., about −16° C. to about −20° C., about −17° C. to about −19° C., or about −18° C. In particular examples, the holding temperature in the primary drying step is between about -17°C and about -19°C, or about -18°C.

[0180] The pressure (vacuum condition) can be any suitable pressure. In some cases, the pressure should be 50% or less of the vapor pressure of ice at the glass transition temperature of the formulation (e.g., about 0.270 mbar). The pressure should not be too low. For example, the pressure can be about 0.140 to about 0.050, about 0.100 to about 0.060, about 0.100 to about 0.070, about 0.100 to about 0.080, about 0.099 to about 0.081, about 0.098 to about 0.082, about 0.097 to about 0.083, about 0.096 to about 0.084, about 0.095 to about 0.085, about 0.094 to about 0.086, about 0.093 to about 0.087, about 0.092 to about 0.088, about 0.091 to about 0.089, about 0.090 mbar, or about 0.120 mbar. In a specific example, the pressure is about 0.090 mbar.

[0181] In a particular example, the holding temperature in the primary drying step is between about −17° C. and about −19° C. (e.g., about −18° C.), the primary drying step involves raising the temperature to the holding temperature at a rate of about 1° C. per minute, and the primary drying step is a step of about 10 hours to about 40 hours (e.g., about 20 to about 40 hours, or about 25 to 35 hours, e.g., about 30 hours or about 32 hours). E. Secondary Drying Step

[0182] The third step in freeze-drying is the secondary drying step, in which the composition containing the bacteria or Listeria strains produced by the primary drying step is exposed to a vacuum at an elevated temperature, during which unfrozen water is removed by desorption.

[0183] For example, the temperature for the secondary drying step can be reached by increasing the temperature (e.g., shelf temperature) at a rate of about 0.2°C to about 2.0°C per minute. Alternatively, the holding temperature for the secondary drying step (e.g., shelf temperature) can be reached by increasing the temperature (e.g., shelf temperature) at a rate of, for example, about 0.2°C to about 1.8°C per minute, about 0.2°C to about 1.6°C per minute, about 0.2°C to about 1.4°C per minute, about 0.2°C to about 1.2°C per minute, about 0.2°C to about 1.0°C per minute, about 0.2°C to about 0.8°C per minute, about 0.2°C to about 0.6°C per minute, or about 0.2°C to about 0.4°C per minute. For example, the temperature can be increased to the secondary drying temperature at a rate of about 0.2°C, about 0.3°C, about 0.4°C, about 0.5°C, about 0.6°C, about 0.7°C, about 0.8°C, about 0.9°C, about 1.0°C, about 1.1°C, about 1.2°C, about 1.3°C, about 1.4°C, about 1.5°C, about 1.6°C, about 1.7°C, about 1.8°C, about 1.9°C, or about 2.0°C per minute. In a particular example, the hold temperature of the secondary drying step is reached by increasing the temperature to the hold temperature at a rate of about 0.2°C per minute.

[0184] The secondary drying step can be of any suitable duration. Similarly, the temperature (e.g., shelf temperature or hold temperature) can be held at the secondary drying temperature for any suitable time. For example, the temperature can be held at the secondary drying temperature for any suitable time to achieve the desired residual moisture level in the freeze-dried product. For example, the secondary drying step can be about 5 to about 40, about 10 to about 30, about 15 to about 25, about 2 to about 25, about 2 to about 20, about 2 to about 10, about 2 to about 4, about 1 to about 25, about 1 to about 20, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1.5 to about 2.5, about 2.5 to about 3.5, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 hour, or the temperature can be held at the secondary drying temperature for the aforementioned time. In certain examples, the secondary drying step can be a 10 hour or less step, or the secondary drying time is 10 hours or less. In another specific example, the secondary drying holding time is 6 hours or less. In another specific example, the temperature can be held at the secondary drying temperature for about 3 hours. In another specific example, the temperature can be held at the secondary drying temperature for about 2 hours. In one example, the secondary drying step is from about 1 hour to about 10 hours. In another example, the secondary drying step includes holding the composition at the holding temperature for about 2 hours to about 6 hours, about 5 hours to about 6 hours, or about 5 hours or about 6 hours.

[0185] The secondary drying temperature (i.e., shelf temperature or holding temperature) can be any temperature suitable for drying the bacteria or Listeria strain to achieve the desired residual moisture level in the lyophilized product. For example, the temperature can be between about 5°C and about 40°C, about 5°C and about 30°C, about 10°C and about 30°C, about 20°C and about 30°C, or about 15°C and about 25°C. In a particular example, the secondary drying temperature can be about 25°C. In another particular example, the secondary drying temperature can be about 20°C. In another particular example, the secondary drying temperature is about 20°C or lower. In another example, the temperature can be between about 5°C and about 20°C, about 9°C and about 15°C, about 10°C and about 15°C, about 11°C and about 14°C, about 11°C and about 13°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, or about 20°C. In a particular example, the secondary drying temperature can be about 12°C. Alternatively, the holding temperature can be between about −10° C. and about 30° C., between about −10° C. and about 25° C., between about −10° C. and about 20° C., between about −10° C. and about 10° C., between about −5° C. and about 30° C., between about −5° C. and about 25° C., between about −5° C. and about 20° C., between about −5° C. and about 15° C., between about −5° C. and about 10° C., between about −5° C. and about 5° C., between about −4° C. and about 4° C., between about −3° C. and about 3° C., between about −2° C. and about 2° C., about −1° C. and about 1° C., or about 0° C. In certain examples, the holding temperature can be between about −5° C. and about 5° C. or about 0° C.

[0186] The pressure (vacuum conditions) can be any suitable pressure. In some cases, the pressure is in the same range as for the primary drying step. However, some cycles during secondary drying may have a full vacuum. For example, the pressure can be about 0.140 to about 0.020, 0.140 to about 0.030, 0.140 to about 0.040, 0.140 to about 0.050, about 0.100 to about 0.060, about 0.100 to about 0.070, about 0.100 to about 0.080, about 0.099 to about 0.081, about 0.098 to about 0.082, about 0.097 to about 0.083, about 0.096 to about 0.084, about 0.095 to about 0.085, about 0.094 to about 0.086, about 0.093 to about 0.087, about 0.092 to about 0.088, about 0.091 to about 0.089, about 0.090 mbar, or about 0.120 mbar. In a particular example, the pressure is about 0.090 mbar.

[0187] In a particular example, the holding temperature in the secondary drying step is between about -5°C and about 5°C (e.g., about 0°C), the secondary drying step includes increasing the temperature to the holding temperature at a rate of about 0.2°C per minute, and the secondary drying step includes holding the composition at the holding temperature for about 5 hours to about 6 hours.

[0188] The secondary drying step can result in a lyophilized product with any desired residual moisture, for example, about 7.0%, about 6.9%, about 6.8%, about 6.7%, about 6.6%, about 6.5%, about 6.4%, about 6.3%, about 6.2%, about 6.1%, about 6.0%, about 5.9%, about 5.8%, about 5.7%, about 5.6%, about 5.5%, about 5.4%, about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%, about 4.1%, or about 4. It can be 0%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3.0%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, or about 1.0% or less. Alternatively, the residual moisture is at least about 7.0%, about 6.9%, about 6.8%, about 6.7%, about 6.6%, about 6.5%, about 6.4%, about 6.3%, about 6.2%, about 6.1%, about 6.0%, about 5.9%, about 5.8%, about 5.7%, about 5.6%, about 5.5%, about 5.4%, about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%, or about 4.1%. , about 4.0%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3.0%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, or about 1.0%. In certain examples, the residual moisture can be at least about 1%, at least about 1.5%, or at least about 2%, and not more than about 7%.Alternatively, the residual moisture can be between about 1% and about 7%, about 1% and about 6.5%, about 1% and about 6%, about 1% and about 5.5%, about 1% and about 5%, about 1.5% and about 7%, about 1.5% and about 6.5%, about 1.5% and about 6%, about 1.5% and about 5.5%, about 1.5% and about 5%, about 1.5% and about 4.5%, about 2% and about 7%, about 2% and about 6.5%, about 2% and about 6%, about 2% and about 5.5%, about 2% and about 5%, about 2% and about 4.5%, about 2% and about 4%, about 2% and about 3%, or about 3% and about 4%. In certain examples, the residual moisture content can be about 3% to about 4%, about 3.1% to about 3.9%, about 3.2% to about 3.8%, about 3.3% to about 3.7%, about 3.4% to about 3.6%, or about 3.5%. In certain examples, the residual moisture content is at least about 2%, at least about 2.5%, or at least about 3%. In other specific examples, the residual moisture content is between about 1% and about 5%, between about 2% and about 4%, between about 2.5% and about 3.5%, between about 2.5% and about 4%, between about 3% and about 4%, or between about 3% and about 3.5%. F. Storage and Reconstitution of Lyophilized Bacteria or Listeria

[0189] The resulting freeze-dried bacteria or Listeria can be a freeze-dried composition containing any combination of the ingredients listed in the formulation section. In one example, the freeze-dried composition contains a Listeria strain, a buffer (e.g., phosphate), and an excipient (e.g., sucrose). Optionally, the freeze-dried composition does not contain one or more or all of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). Optionally, the freeze-dried composition does not contain one or more or all of the optional ingredients listed in the formulation section.

[0190] The resulting lyophilized bacteria or Listeria may be a lyophilized composition having any of the residual moisture levels listed elsewhere herein. In one example, the residual moisture level may be between about 1% and about 5%, between about 2% and about 4%, or between about 3% and about 4%.

[0191] Freeze-dried bacteria can be stored under any suitable conditions, including any suitable temperature, relative humidity, and atmospheric oxygen level, and these conditions are well known. After a defined period of storage, the freeze-dried bacteria or Listeria can exhibit at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% viability upon reconstitution. Reconstitution can occur after storage of the freeze-dried bacteria or Listeria for, for example, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, 12 months (1 year), 15 months, 18 months, 21 months, or 24 months (2 years).

[0192] The storage temperature for freeze-dried bacteria or Listeria can be, for example, between about 0°C and about 10°C, about 1°C and about 9°C, about 2°C and about 8°C, about 2°C and about 6°C, or about 3°C ​​and about 5°C. In particular examples, the storage temperature can be between about 2°C and about 8°C, or the storage temperature can be about 4°C. In other examples, the storage temperature can be between about -15°C and about -25°C, about -16°C and about -24°C, about -17°C and about -23°C, about -18°C and about -22°C, or about -19°C and about -21°C. In particular examples, the storage temperature can be about -20°C.

[0193] For example, the freeze-dried bacteria or Listeria may exhibit a viability of at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% after storage at about 2-8°C (e.g., 4°C) or about -20°C for about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months. The freeze-dried bacteria or Listeria may exhibit a viability of at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% after storage at about 30°C, at about room temperature (i.e., about 20-25°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C), at about 2-8°C (e.g., 4°C), or about -20°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, or about 24 months. By way of example, the freeze-dried bacteria or Listeria may exhibit at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% after storage at about 30°C, at about room temperature (i.e., about 20-25°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C), at about 2-8°C (e.g., 4°C), or about -20°C. As another example, freeze-dried bacteria or Listeria may exhibit at least about 75% to about 80% viability after 9 months at -20°C. As another example, freeze-dried bacteria or Listeria may exhibit at least about 95% to about 100% viability after 9 months at -20°C. As another example, freeze-dried bacteria or Listeria may exhibit at least about 80% to about 90% viability after 2 months at room temperature or at 30°C. As another example, freeze-dried bacteria or Listeria may exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%, or 90% viability after about 12 months, 18 months, or 24 months at about -20°C. As another example, freeze-dried bacteria or Listeria may exhibit at least about 60%, 65%, 70%, 75%, or 80% viability after about 12 months, 18 months, or 24 months at about 2-8°C.As another example, the freeze-dried bacteria or Listeria may exhibit at least about 60%, 65%, 70%, 75%, or 80% viability after about 12 months, 18 months, or 24 months at about 2-8°C.

[0194] After storage, the freeze-dried bacteria or Listeria strains can be reconstituted with a solvent or diluent (e.g., water) as needed. In one example, the solvent or diluent can be a suitable medium for culturing the bacteria or Listeria strains. Methods for reconstituting and rehydrating freeze-dried bacteria or Listeria strains are well known. In one example, the volume of solvent used is the volume of the pre-lyophilization solution used to create the freeze-dried bacteria or Listeria strains. In another example, the volume of solvent used is greater than the volume of the pre-lyophilization solution used to create the freeze-dried bacteria or Listeria strains. In another example, the volume of solvent used is less than the volume of the pre-lyophilization solution used to create the freeze-dried bacteria or Listeria strains.

[0195] The reconstitution time can be any suitable reconstitution time. For example, the reconstitution time can be less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 minutes. In a particular example, the reconstitution time is less than about 2 minutes. III. Recombinant Bacteria or Listeria Strains

[0196] The lyophilized compositions disclosed herein and compositions that undergo the lyophilization methods disclosed herein contain bacterial strains, e.g., Listeria strains. Such bacterial strains may be recombinant bacterial strains. Such recombinant bacterial strains may contain recombinant fusion polypeptides disclosed herein or may contain nucleic acids encoding the recombinant fusion polypeptides as disclosed elsewhere herein. In some embodiments, the bacterial strain is a Listeria strain, e.g., a Listeria monocytogenes (Lm) strain. Lm has many inherent advantages as a vaccine vector. The bacterium grows very efficiently in vitro without special requirements, and it lacks LPS, a major virulence factor in Gram-negative bacteria such as Salmonella. Genetically attenuated Lm vectors allow for easy removal with antibiotics if severe adverse effects occur and also provide additional safety because, unlike some viral vectors, genetic material does not integrate into the host genome.

[0197] The recombinant Listeria strain can be any Listeria strain. Examples of suitable Listeria strains include Listeria seeligeri, Listeria grayi, Listeria ivanovii, Listeria murrayi, Listeria welshimeri, Listeria monocytogenes (Lm), or any other known Listeria species. In some embodiments, the recombinant Listeria strain is a strain of the Listeria monocytogenes species. Examples of Listeria monocytogenes strains include: L. monocytogenes 10403S wild-type (see, e.g., Bishop and Hinrichs (1987) J Immunol 139:2005-2009; Lauer et al. (2002) J Bact 184:4177-4186); phage-cured L. monocytogenes DP-L4056 (see, e.g., Lauer et al. (2002) J Bact 184:4177-4186); phage-cured L. monocytogenes DP-L4027 with an hly gene deletion (see, e.g., Lauer et al. (2002) J Bact 184:4177-4186; Jones and Portnoy (1994) Infect Immunity 65:5608-5613); L. monocytogenes DP-L4029, which has been phage-cured and has an actA gene deletion (see, e.g., Lauer et al. (2002) J Bact 184:4177-4186; Skoble et al. (2000) J Cell Biol 150:527- 538); L. monocytogenes DP-L4042 (Delta PEST) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci. USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4097 (LLO-S44A) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4364 (delta lplA; lipoic acid protein ligase) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4405 (delta inlA) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4406 (delta inlB) (see, e.g., Brockstedt et al. al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes CS-LOOOl (delta actA; delta lplB) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes CS-L0002 (delta actA; delta lplA) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information). USA 101:13832-13837 and supporting information); L. monocytogenes CS-L0003 (LLO L461T; delta lplA) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4038 (delta actA; LLO L461T) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes DP-L4384 (LLO S44A; LLO L461T) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L. monocytogenes strains with a deletion of lplA1 (encoding the lipoic acid protein ligase LplA1) (see, e.g., O'Riordan et al. (2003) Science 302:462-464); L. monocytogenes DP-L4017 (10403S with LLO L461T) (see, e.g., US 7,691,393); L. monocytogenes EGD (see, e.g., GenBank Accession No. AL591824). In another embodiment, the Listeria strain is L. monocytogenes EGD-e (see, e.g., GenBank Accession No. NC_003210; ATCC Accession No. BAA-679); L. monocytogenes DP-L4029 (actA deletion, optionally with uvrAB deletion) (DP-L4029uvrAB) (see, e.g., US 7,691,393); L. monocytogenes actA- / inlB- double mutant (see, e.g., ATCC Accession No. PTA-5562); L. monocytogenes lplA mutant or hly mutant (see, e.g., US 2004 / 0013690); L. monocytogenes dal / dat double mutant (see, e.g., US 2005 / 0048081).Other L. monocytogenes strains include those that have been modified (e.g., by plasmid and / or genomic integration) to contain a nucleic acid encoding one or any combination of the following genes: hly (LLO; listeriolysin), iap (p60), inlA, inlB, inlC, dal (alanine racemase), dat (D-amino acid aminotransferase), plcA, plcB, actA; or any nucleic acid that mediates unilamellar vesicle growth, spreading, degradation, bilamellar vesicle degradation, binding to or uptake by host cells. Each of the above references is incorporated herein by reference in its entirety and for all purposes.

[0198] The recombinant bacterium or Listeria may have wild-type virulence, attenuated virulence, or no virulence. For example, the recombinant Listeria may be virulent enough to escape the phagosome or phagolysosome and enter the cytosol. Such Listeria strains may be live-attenuated Listeria strains containing at least one attenuating mutation, deletion, or inactivation as disclosed elsewhere herein. In some embodiments, the recombinant Listeria is an attenuated auxotrophic strain. An auxotrophic strain is one that cannot synthesize a particular organic compound required for its growth. Examples of such strains are described in U.S. Pat. No. 8,114,414, the entire contents of which are incorporated herein by reference for all purposes.

[0199] In some embodiments, the recombinant Listeria strain lacks an antibiotic resistance gene. For example, such a recombinant Listeria strain may contain a plasmid that does not encode an antibiotic resistance gene. However, some recombinant Listeria strains provided herein contain a plasmid containing a nucleic acid encoding an antibiotic resistance gene. The antibiotic resistance gene can be used in conventional selection and cloning processes commonly utilized in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin. A. Bacterial or Listeria Strains Containing Recombinant Fusion Polypeptides or Containing Nucleic Acids Encoding Recombinant Fusion Polypeptides

[0200] The recombinant bacterial strains (e.g., Listeria strains) disclosed herein comprise a recombinant fusion polypeptide disclosed herein or comprise a nucleic acid encoding a recombinant fusion polypeptide as disclosed elsewhere herein.

[0201] In bacteria or Listeria strains containing nucleic acids encoding recombinant fusion proteins, the nucleic acids can be codon-optimized. Examples of optimal codons used by L. monocytogenes for each amino acid are shown in US2007 / 0207170, the entire contents of which are incorporated herein by reference for all purposes. A nucleic acid is codon-optimized when at least one codon in the nucleic acid is replaced with a codon that is more frequently used by L. monocytogenes for that amino acid compared to that codon in the original sequence.

[0202] The nucleic acids may be present on an episomal plasmid in the bacterium or Listeria strain and / or the nucleic acids may be integrated into the genome of the bacterium or Listeria strain. Some recombinant bacteria or Listeria strains contain two separate nucleic acids encoding the two recombinant fusion polypeptides disclosed herein: one nucleic acid in an episomal plasmid and one nucleic acid integrated into the genome of the bacterium or Listeria strain.

[0203] Episomal plasmids can be stably maintained in vitro (in cell culture), in vivo (within a host), or both in vitro and in vivo. When in an episomal plasmid, the open reading frame encoding the recombinant fusion polypeptide may be operably linked to a promoter / regulatory sequence in the plasmid. When integrated into the genome of a bacterium or Listeria strain, the open reading frame encoding the recombinant fusion polypeptide may be operably linked to an exogenous promoter / regulatory sequence or to an endogenous promoter / regulatory sequence. Examples of promoter / regulatory sequences useful for driving constitutive expression of genes are well known and include, for example, the Listeria hly, hlyA, actA, prfA, and p60 promoters, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter. In some cases, the inserted gene of interest is not disrupted or subject to the regulatory constraints that often result from integration into genomic DNA, and in some cases, the presence of the inserted heterologous gene does not result in rearrangement or disruption of significant regions of the cell itself.

[0204] Such recombinant bacteria or Listeria strains can be produced by transforming bacteria or Listeria strains, or attenuated bacteria or Listeria strains described elsewhere herein, with a plasmid or vector containing a nucleic acid encoding the recombinant fusion polypeptide. The plasmid may be an episomal plasmid that does not integrate into the host chromosome. Alternatively, the plasmid may be an integrative plasmid that integrates into the chromosome of the bacterium or Listeria strain. Plasmids used herein may also be multicopy plasmids. Methods for transforming bacteria are well known and include calcium chloride competent cell-based methods, electroporation, bacteriophage-mediated transduction, chemical transformation techniques, and physical transformation techniques. See, e.g., de Boer et al. (1989) Cell 56:641-649; Miller et al. (1995) FASEB J. 9:190-199; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al. (1997) Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhard et al. al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC; and Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, each of which references is incorporated herein by reference in its entirety for all purposes.

[0205] Bacteria or Listeria strains with heterologous nucleic acids integrated into their genomes can be produced, for example, by using a site-specific integration vector, whereby bacteria or Listeria containing the integrated gene are generated using homologous recombination. The integration vector can be any site-specific integration vector capable of infecting bacteria or Listeria strains. Such integration vectors can include, for example, a PSA attPP' site, a gene encoding PSA integrase, a U153 attPP' site, a gene encoding U153 integrase, an A118 attPP' site, a gene encoding A118 integrase, or any other known attPP' site, or any other phage integrase.

[0206] Such bacteria or Listeria strains containing the integrated gene can also be produced using any other known method for integrating heterologous nucleic acids into bacterial or Listeria chromosomes. The technique of homologous recombination is well known and is described, for example, in Baloglu et al. (2005) Vet Microbiol 109(1-2):11-17; Jiang et al. 2005) Acta Biochim Biophys Sin (Shanghai) 37(1):19-24), and US 6,855,320, each of which is incorporated herein by reference in its entirety for all purposes.

[0207] Integration into the chromosome of bacteria or Listeria can also be achieved by using transposon insertion.Transposon insertion technology is well known, for example, the construction of DP-L967 is described by Sun et al. (1990) Infection and Immunity 58: 3770-3778, the entire contents of which are incorporated herein by reference for all purposes.Transposon mutagenesis can achieve stable genome insertion, but the position in the genome where heterologous nucleic acid is inserted is unknown.

[0208] Integration into the bacterial or Listeria chromosome can also be achieved using phage integration sites (see, e.g., Lauer et al. (2002) J Bacteriol 184(15):4177-4186, incorporated herein by reference in its entirety for all purposes). For example, the integrase gene and attachment site of a bacteriophage (e.g., U153 or PSA listeriophage) can be used to insert a heterologous gene into the corresponding attachment site, which can be any suitable site within the genome (e.g., comK or the 3' end of the arg tRNA gene). The endogenous prophage can be cured from the attachment site to be utilized prior to integration of the heterologous nucleic acid. Such methods can, for example, result in single-copy integrants. To avoid the "phage curing step", a phage integration system based on PSA phage can be used (see, for example, Lauer et al. (2002) J Bacteriol 184:4177-4186, the entire contents of which are incorporated herein by reference for all purposes). Maintenance of the integrated gene may require continuous selection, for example, with an antibiotic. Alternatively, a phage-based chromosomal integration system can be established that does not require antibiotic selection. Instead, an auxotrophic host strain can be complemented. For example, a phage-based chromosomal integration system for clinical applications can be used, in which case a host strain (e.g., Lm dal(-)dat(-)) that is auxotrophic for essential enzymes, such as D-alanine racemase, is used.

[0209] Conjugation can also be used to introduce genetic material and / or plasmid into bacteria.Conjugation methods are well known, and are described, for example, in Nikodinovic et al. (2006) Plasmid 56(3):223-227 and Auchtung et al. (2005) Proc Natl Acad Sci USA 102(35):12554-12559, and each of these references is incorporated herein by reference in its entirety for all purposes.

[0210] In certain examples, a recombinant bacterium or Listeria strain can contain a nucleic acid encoding a recombinant fusion polypeptide integrated into the bacterial or Listeria genome as an open reading frame with the endogenous actA sequence (encoding the ActA protein) or the endogenous hly sequence (encoding the LLO protein). For example, expression and secretion of the fusion polypeptide can be under the control of the endogenous actA promoter and ActA signal sequence, or under the control of the endogenous hly promoter and LLO signal sequence. In another example, the nucleic acid encoding the recombinant fusion polypeptide can replace the actA sequence encoding the ActA protein or the hly sequence encoding the LLO protein.

[0211] Selection of recombinant bacteria or Listeria strains can be achieved by any means. For example, antibiotic selection can be used. Antibiotic resistance genes can be used in conventional selection and cloning processes commonly utilized in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin. Alternatively, auxotrophic strains can be used, and exogenous metabolic genes can be used for selection instead of or in addition to antibiotic resistance genes. As an example, to select for auxotrophic bacteria containing a plasmid encoding a metabolic enzyme or complementary gene provided herein, the transformed auxotrophic bacteria can be grown in a medium that selects for the expression of the metabolic enzyme-encoding gene (e.g., an amino acid metabolism-related gene) or complementary gene. Alternatively, temperature-sensitive plasmids can be used to select for recombinants, or any other known means can be used to select for recombinants. B. Attenuation of bacteria or Listeria strains

[0212] The recombinant bacterial strains (e.g., recombinant Listeria strains) disclosed herein can be attenuated. The term "attenuation" encompasses a reduction in the ability of the bacteria to cause disease in a host animal. For example, attenuated Listeria can be grown and maintained in culture, but the pathogenic properties of the attenuated Listeria strain can be reduced compared to wild-type Listeria. In some embodiments, using intravenous inoculation of attenuated Listeria into BALB / c mice as an example, the lethal dose at which 50% of the inoculated animals survive (LD 50 ) is the LD of wild-type Listeria 50The attenuated Listeria strain is increased by at least about 10-fold, at least about 100-fold, at least about 1,000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the wild-type, non-attenuated strain. Thus, an attenuated strain of Listeria is either non-lethal to the animal to which it is administered, or is lethal to the animal only when the number of bacteria administered is significantly greater than the number of wild-type, non-attenuated bacteria required to kill the same animal. Attenuated bacteria should also be construed to mean one that is unable to replicate in the general environment because the nutrients necessary for its growth are not present there. Thus, the bacteria are limited to replicating in a controlled environment where the necessary nutrients are provided. Attenuated strains are environmentally safe because they are incapable of uncontrolled replication. (1) Methods for attenuating bacteria and Listeria strains

[0213] Attenuation can be achieved by any known means. For example, such attenuated strains can be deficient in one or more endogenous virulence genes or in one or more endogenous metabolic genes. Examples of such genes are disclosed herein, and attenuation can be achieved by inactivation of any one or any combination of the genes disclosed herein. Inactivation can be achieved, for example, by deletion or by mutation (e.g., inactivating mutation). The term "mutation" includes any type of mutation or modification to a sequence (nucleic acid or amino acid sequence) and can encompass deletion, truncation, insertion, substitution, disruption, or translocation. For example, mutations can include frameshift mutations, mutations that cause premature termination of a protein, or mutations in regulatory sequences that affect gene expression. Mutagenesis can be achieved using recombinant DNA technology or traditional mutagenesis techniques using mutagenic chemicals or radiation, followed by selection of mutants. In some embodiments, deletion mutants are used because of the concomitant low probability of reversion. The term "metabolic gene" refers to a gene encoding an enzyme involved in or required for the synthesis of a nutrient utilized or required by the host bacterium. For example, the enzyme may be involved in or required for the synthesis of a nutrient necessary for the sustained growth of the host bacterium. The term "virulence" gene includes genes whose presence or activity in the genome of an organism contributes to the pathogenicity of the organism (e.g., enabling the organism to colonize a niche in the host (including adhering to cells), immune evasion (evading the host's immune response), immune suppression (inhibiting the host's immune response), invade and exit cells, or obtain nutrients from the host).

[0214] A specific example of such an attenuated strain is Listeria monocytogenes (Lm) dal(-) dat(-) (Lmdd). Another example of such an attenuated strain is Lm dal(-) dat(-) ΔactA (LmddA). See, e.g., US2011 / 0142791, incorporated herein by reference in its entirety for all purposes. LmddA is based on a Listeria strain that is attenuated due to deletion of the endogenous virulence gene actA. Such strains can harbor plasmids for in vivo and in vitro antigen expression through complementation of the dal gene. Alternatively, LmddA can be a dal / dat / actA Listeria with mutations in the endogenous dal, dat, and actA genes. Such mutations can be, for example, deletions or other inactivating mutations.

[0215] Another specific example of an attenuated strain is Lm prfA(-), or a strain with a partial deletion or inactivating mutation of the prfA gene. The PrfA protein controls the expression of a regulon containing essential virulence genes required for Lm to colonize its vertebrate host. Thus, a prfA mutation severely impairs the ability of PrfA to activate the expression of PrfA-dependent virulence genes.

[0216] Yet another specific example of an attenuated strain is Lm inlB(-)actA(-), in which two genes important for the natural virulence of the bacterium - internalin B and actA - are deleted.

[0217] Other examples of attenuated bacteria or Listeria strains include bacteria or Listeria strains that are deficient in one or more endogenous virulence genes. Examples of such genes include actA, prfA, plcB, plcA, inlA, inlB, inlC, inlJ, and bsh in Listeria. Attenuated Listeria strains can also be double or triple mutants of any of the above-mentioned strains. Attenuated Listeria strains can contain mutations or deletions in each gene of the genes provided herein (including, for example, the actA, prfA, and dal / dat genes), or can contain mutations or deletions in, for example, 10 or fewer of any of the above genes. For example, attenuated Listeria strains can contain mutations or deletions in the endogenous internalin C (inlC) gene and / or mutations or deletions in the endogenous actA gene. Alternatively, the attenuated Listeria strain may contain a mutation or deletion of the endogenous internalin B (inlB) gene and / or a mutation or deletion of the endogenous actA gene. Alternatively, the attenuated Listeria strain may contain a mutation or deletion of the endogenous inlB, inlC, and actA genes. Translocation of Listeria to neighboring cells is inhibited by deletion of the endogenous actA gene and / or the endogenous inlC gene or the endogenous inlB gene, which are involved in that process, thereby resulting in a high level of attenuation, as well as increased immunogenicity and usefulness as a strain backbone. The attenuated Listeria strain may also be a double mutant containing a mutation or deletion of both plcA and plcB. In some cases, strains may be constructed from an EGD Listeria backbone.

[0218] The bacteria or Listeria strain may be an auxotrophic strain with a mutation in a metabolic gene. For example, the strain may be deficient in one or more endogenous amino acid metabolism-related genes. For example, the generation of an auxotrophic Listeria strain deficient in D-alanine can be achieved by a number of well-known methods, including deletion mutations, insertion mutations, frameshift mutations, mutations that cause premature termination of proteins, or mutations in regulatory sequences that affect gene expression. In some embodiments, deletion mutants are used because of the low probability of concomitant reversion of the auxotrophic phenotype. For example, D-alanine mutants generated according to the protocol presented herein can be tested for their ability to grow in the absence of D-alanine in a simple laboratory culture assay. Mutants that cannot grow in the absence of this compound can be selected.

[0219] Examples of endogenous amino acid metabolism-related genes include vitamin synthesis genes, genes encoding pantothenate synthase, D-glutamate synthase, D-alanine aminotransferase (dat), D-alanine racemase (dal), dga, genes involved in the synthesis of diaminopimelic acid (DAP), genes involved in the synthesis of cysteine ​​synthase A (cysK), vitamin B12-dependent methionine synthase, trpA, trpB, trpE, asnB, gltD, gltB, leuA, argG, and thrC. Listeria strains may be defective in two or more such genes (e.g., dat and dal). D-glutamate synthesis is controlled in part by the dal gene, which is involved in the conversion of D-glupyr to alpha-ketoglutarate and D-ala, and the reverse reaction.

[0220] As another example, attenuated Listeria strains may be deficient in endogenous synthase genes, such as amino acid synthesis genes, including folP, genes encoding dihydrouridine synthase family proteins, ispD, ispF, genes encoding phosphoenolpyruvate synthase, hisF, hisH, fliI, genes encoding large ribosomal subunit pseudouridine synthase, ispD, genes encoding bifunctional GMP synthase / glutamine amidotransferase proteins, cobS, cobB, cbiD, and uroporphyrin-III. C-methyltransferase / uroporphyrinogen-III synthase-encoding gene, cobQ, uppS, truB, dxs, mvaS, dapA, ispG, folC, citrate synthase-encoding gene, argJ, 3-deoxy-7-phosphoheptulosonate synthase-encoding gene, indole-3-glycerol phosphate synthase-encoding gene, anthranilate synthase / glutamine amidotransferase component-encoding gene, menB, menaquinone-specific isochorismate synthase Genes encoding the ATP synthases include genes encoding the ATP synthases ATP-1, ATP-2, ATP-3, ATP-4, ATP-5, ATP-6, ATP-7, ATP-8, ATP-9, ATP-10, ATP-11, ATP-12, ATP-13, ATP-14, ATP-15, ATP-16, ATP-17, ATP-18, ATP-19, ATP-20, ATP-21, ATP-22, ATP-23, ATP-24, ATP-25, ATP-26, ATP-27, ATP-28, ATP-29, ATP-29, ATP-21, ATP-22, ATP-23, ATP-24, ATP-25, ATP-26, ATP-27, ATP-28, ATP-29, ATP-29, ATP-29, ATP-29, ATP-29, ATP-20, ATP-21, ATP-22, ATP-23, ATP-24, ATP-25, ATP-26, ATP-27, ATP-28 ...

[0221] Attenuated Listeria strains can also be deficient in endogenous phoP, aroA, aroC, aroD, or plcB. In yet another example, attenuated Listeria strains can be deficient in endogenous peptide transporters, including ABC transporter / ATP-binding / permease protein, oligopeptide ABC transporter / oligopeptide-binding protein, oligopeptide ABC transporter / permease protein, zinc ABC transporter / zinc-binding protein, sugar ABC transporter, phosphate transporter, ZIP zinc transporter, EmrB / QacA family drug resistance transporters, sulfate transporter, proton-dependent oligopeptide transporter, magnesium transporter, folate / nitrate transporter, spermidine / putrescine ABC transporter, Na / Pi symporter, sugar phosphate transporter, glutamine ABC transporter, major facilitator family transporters, glycine betaine / L-proline ABC transporter, molybdenum ABC transporter, teichoic acid (techoic acid) transporter, and the like. acid ABC transporter, cobalt ABC transporter, ammonium transporter, amino acid ABC transporter, cell division ABC transporter, manganese ABC transporter, iron compound ABC transporter, maltose / maltodextrin ABC transporter, drug resistance transporter of the Bcr / CflA family, and genes encoding subunits for one of the above proteins.

[0222] Other attenuated bacteria and Listeria strains may lack endogenous metabolic enzymes that metabolize amino acids used in bacterial growth, replication, cell wall synthesis, protein synthesis, fatty acid metabolism, or any other growth or replication process. Similarly, attenuated strains may lack endogenous metabolic enzymes that can catalyze the formation of amino acids used in cell wall synthesis, endogenous metabolic enzymes that can catalyze the synthesis of amino acids used in cell wall synthesis, or endogenous metabolic enzymes that can be involved in the synthesis of amino acids used in cell wall synthesis. Alternatively, amino acids may be used in cell wall biosynthesis. Alternatively, the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.

[0223] Other attenuated Listeria strains may be deficient in metabolic enzymes encoded by the D-glutamate synthesis genes, dga, and alr (alanine racemase) genes, or any other enzyme involved in alanine synthesis. Further examples of metabolic enzymes that may be deficient in Listeria strains include the enzyme encoded by serC (phosphoserine aminotransferase), the enzyme encoded by asd (aspartate beta-semialdehyde dehydrogenase;The enzymes encoded by the gene encoding gsaB - glutamate-1-semialdehyde aminotransferase (catalyzes the formation of 5-aminolevulinate from (S)-4-amino-5-oxopentanoate), the enzyme encoded by hemL (catalyzes the formation of 5-aminolevulinate from (S)-4-amino-5-oxopentanoate), the enzyme encoded by aspB (catalyzes the formation of oxaloacetate (ox) from L-aspartate and 2-oxoglutarate), and the enzyme encoded by α-aspartate (catalyzes the formation of 5-aminolevulinate from L-aspartate and 2-oxoglutarate). aspartate aminotransferase catalyzing the formation of L-alozcetate and L-glutamate), an enzyme encoded by argF-1 (involved in arginine biosynthesis), an enzyme encoded by aroE (involved in amino acid biosynthesis), an enzyme encoded by aroB (involved in 3-dehydroquinate biosynthesis), an enzyme encoded by aroD (involved in amino acid biosynthesis), an enzyme encoded by aroC (involved in amino acid biosynthesis), an enzyme encoded by hisB (involved in histidine biosynthesis) , enzymes encoded by hisD (involved in histidine biosynthesis), enzymes encoded by hisG (involved in histidine biosynthesis), enzymes encoded by metX (involved in methionine biosynthesis), enzymes encoded by proB (involved in proline biosynthesis), enzymes encoded by argR (involved in arginine biosynthesis), enzymes encoded by argJ (involved in arginine biosynthesis), thil (involved in thiamine biosynthesis), enzymes encoded by LMOf2365_1652 (involved in tryptophan biosynthesis) These include the enzymes encoded by aroA (involved in tryptophan biosynthesis), aroB (involved in tryptophan biosynthesis), ilvD (involved in valine and isoleucine biosynthesis), ilvC (involved in valine and isoleucine biosynthesis), leuA (involved in leucine biosynthesis), dapF (involved in lysine biosynthesis), and thrB (involved in threonine biosynthesis) (all GenBank accession number NC_002973);

[0224] Attenuated Listeria strains can also be generated by mutations in other metabolic enzymes, such as tRNA synthetase. For example, the metabolic enzyme can be encoded by the trpS gene, which encodes tryptophanyl-tRNA synthetase. For example, the host strain bacterium can be Δ(trpS aroA), and both markers can be contained on an integration vector.

[0225] Other examples of metabolic enzymes that can be mutated to generate attenuated Listeria strains include the enzyme encoded by murE (involved in the synthesis of diaminopimelic acid; GenBank accession number NC_003485), the enzyme encoded by LMOf2365_2494 (involved in teichoic acid biosynthesis), the enzyme encoded by WecE (lipopolysaccharide biosynthesis protein rffA; GenBank accession number AE014075.1), or the enzyme encoded by amiA (N-acetylmuramoyl-L-alanine amidase). Still other examples of metabolic enzymes include aspartate aminotransferase, histidinol phosphate aminotransferase (GenBank accession number NP_466347), or the cell wall teichoic acid glycosylation protein GtcA.

[0226] Other examples of metabolic enzymes that can be mutated to generate attenuated Listeria strains include enzymes that synthesize peptidoglycan components or precursors, such as UDP-N-acetylmuramylpentapeptide, UDP-N-acetylglucosamine, MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol, GlcNAc-p-(1,4)-MurNAc-(pentapeptide)-pyrophosphorylundecaprenol, or any other peptidoglycan component or precursor.

[0227] Further examples of metabolic enzymes that can be mutated to generate attenuated Listeria strains include the metabolic enzymes encoded by murG, murD, murA-1, or murA-2 (all listed in GenBank accession number NC_002973). Alternatively, the metabolic enzyme can be any other synthetic enzyme of a peptidoglycan component or precursor. The metabolic enzyme can also be a trans-glycosylase, trans-peptidase, carboxy-peptidase, any other class of metabolic enzyme, or any other metabolic enzyme. For example, the metabolic enzyme can be any other Listeria metabolic enzyme or any other Listeria monocytogenes metabolic enzyme.

[0228] Other bacterial strains can be attenuated as described above for Listeria by mutating the corresponding orthologous genes in the other bacterial strains. (2) Methods for Complementing Attenuated Bacteria and Listeria Strains

[0229] The attenuated bacteria or Listeria strains disclosed herein can further comprise a nucleic acid comprising a complementary gene or encoding a metabolic enzyme that complements the attenuating mutation (e.g., complements the auxotrophy of an auxotrophic Listeria strain). For example, a nucleic acid having a first open reading frame encoding a fusion polypeptide as disclosed herein can further comprise a second open reading frame comprising a complementary gene or encoding the complementary metabolic enzyme. Alternatively, a first nucleic acid can encode the fusion polypeptide, and a separate, second nucleic acid can comprise the complementary gene or encode the complementary metabolic enzyme.

[0230] The complementary gene may be extrachromosomal or integrated into the bacterial or Listeria genome. For example, an auxotrophic Listeria strain may contain an episomal plasmid containing a nucleic acid encoding a metabolic enzyme. Such a plasmid would be contained in the Listeria in an episomal or extrachromosomal manner. Alternatively, an auxotrophic Listeria strain may contain an integrative plasmid (i.e., an integrative vector) containing a nucleic acid encoding a metabolic enzyme. Such an integrative plasmid can be used for integration into the Listeria chromosome. In some embodiments, the episomal or integrative plasmid lacks an antibiotic resistance marker.

[0231] Instead of or in addition to antibiotic resistance genes, metabolic genes can be used for selection. As an example, to select for auxotrophic bacteria containing a plasmid encoding a metabolic enzyme or complementary gene provided herein, the transformed auxotrophic bacteria can be grown in a medium that selects for the expression of a gene encoding a metabolic enzyme (e.g., an amino acid metabolism-related gene) or complementary gene. For example, a bacterium auxotrophic for D-glutamic acid synthesis can be transformed with a plasmid containing a gene for D-glutamic acid synthesis; the auxotrophic bacterium will grow in the absence of D-glutamic acid, while auxotrophic bacteria not transformed with the plasmid or those not expressing a plasmid encoding a protein for D-glutamic acid synthesis will not grow. Similarly, a bacterium auxotrophic for D-alanine synthesis will grow in the absence of D-alanine when transformed and expressing a plasmid containing a nucleic acid encoding an amino acid metabolic enzyme for D-alanine synthesis. Such methods for producing suitable media containing or lacking necessary growth factors, nutritional supplements, amino acids, vitamins, antibiotics, etc. are well known and commercially available.

[0232] Once auxotrophic bacteria containing the plasmids encoding the metabolic enzymes or complementing genes provided herein have been selected on an appropriate medium, the bacteria can be propagated under selective pressure. Such propagation can include growing the bacteria in a medium that does not contain the auxotrophic factor. The presence of the plasmid expressing the metabolic enzyme or complementing gene in the auxotrophic bacteria ensures that the plasmid replicates with the bacteria, thus resulting in continuous selection of bacteria carrying the plasmid. Production of the bacteria or Listeria strains can be easily scaled up by adjusting the volume of the medium in which the auxotrophic bacteria containing the plasmid are grown.

[0233] In one particular example, the attenuated strain is a strain with a deletion or inactivating mutation in dal and dat (e.g., Listeria monocytogenes (Lm) dal(-) dat(-) (Lmdd) or Lm dal(-) dat(-) ΔactA (LmddA)), and the complementing gene encodes an alanine racemase enzyme (e.g., encoded by the dal gene) or a D-amino acid aminotransferase enzyme (e.g., encoded by the dat gene). An exemplary alanine racemase protein can have the sequence set forth in SEQ ID NO: 76 (encoded by SEQ ID NO: 78; GenBank Accession No. AF038438), or can be a homolog, variant, isoform, analog, fragment, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 76. The alanine racemase protein can also be any other Listeria alanine racemase protein. Alternatively, the alanine racemase protein can be any other Gram-positive alanine racemase protein or any other alanine racemase protein. An exemplary D-amino acid aminotransferase protein can have the sequence set forth in SEQ ID NO: 77 (encoded by SEQ ID NO: 79; GenBank Accession No. AF038439), or can be a homolog, variant, isoform, analog, fragment, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 77. The D-amino acid aminotransferase protein can also be any other Listeria D-amino acid aminotransferase protein. Alternatively, the D-amino acid aminotransferase protein can be any other Gram-positive D-amino acid aminotransferase protein or any other D-amino acid aminotransferase protein.

[0234] In another specific example, the attenuated strain is a strain with a deletion or inactivating mutation in prfA (e.g., Lm prfA(-)), and the complementing gene encodes a PrfA protein. For example, the complementing gene may encode a mutant PrfA(D133V) protein that restores some PrfA function. An example of a wild-type PrfA protein is set forth in SEQ ID NO: 80 (encoded by the nucleic acid sequence set forth in SEQ ID NO: 81), and an example of a D133V mutant PrfA protein is set forth in SEQ ID NO: 82 (encoded by the nucleic acid set forth in SEQ ID NO: 83). The complementing PrfA protein may also be a homolog, variant, isoform, analog, fragment, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 80 or 82. The PrfA protein may also be any other Listeria PrfA protein. Alternatively, the PrfA protein may be any other Gram-positive PrfA protein or any other PrfA protein.

[0235] In another example, the strain or Listeria strain may contain a deletion or inactivating mutation in the actA gene, and the complementing gene may contain the actA gene to complement the mutation and restore function to the Listeria strain.

[0236] Other auxotrophs and complementation systems can also be employed for use with the methods and compositions provided herein. IV. Recombinant Fusion Polypeptides

[0237] The recombinant fusion polypeptides in the recombinant bacteria or Listeria strains disclosed herein can be in any form. Some such fusion polypeptides can contain a PEST-containing peptide fused to one or more disease-associated antigenic peptides. Other such recombinant fusion polypeptides can contain one or more disease-associated antigenic peptides, and the fusion polypeptide does not contain a PEST-containing peptide.

[0238] Another example of a recombinant fusion polypeptide contains, from N- to C-terminus, a bacterial secretory sequence, a ubiquitin (Ub) protein, and one or more disease-associated antigenic peptides (i.e., in tandem, e.g., Ub-peptide1-peptide2). Alternatively, when two or more disease-associated antigenic peptides are used, a combination of separate fusion polypeptides may be used, in which each antigenic peptide is fused to its own secretory sequence and Ub protein (e.g., Ub1-peptide1; Ub2-peptide2).

[0239] Nucleic acids encoding such recombinant fusion polypeptides (referred to as minigene constructs) are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by Shine-Dalgarno ribosome binding site nucleic acid sequences between each open reading frame. For example, the minigene nucleic acid construct can further comprise two to four open reading frames linked by Shine-Dalgarno ribosome binding site nucleic acid sequences between each open reading frame. Each open reading frame can encode a different peptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide can be followed by two stop codons to ensure termination of protein synthesis.

[0240] The bacterial signal sequence can be a Listeria signal sequence, such as an Hly or ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. An exemplary LLO signal sequence is set forth in SEQ ID NO: 97. The signal sequence can be bacterial, native to the host bacterium (e.g., Listeria monocytogenes, e.g., secA1 signal peptide), or foreign to the host bacterium. Specific examples of signal peptides include the Usp45 signal peptide from Lactococcus lactis; the protective antigen signal peptide from Bacillus anthracis; the secA2 signal peptide, such as the p60 signal peptide from Listeria monocytogenes; and the Tat signal peptide, such as the B. subtilis Tat signal peptide (e.g., PhoD). In particular examples, the secretory signal sequence is derived from a Listeria protein, e.g., ActA. 300 secretion signal, or ActA 100 An exemplary ActA signal sequence is set forth in SEQ ID NO:98.

[0241] Ubiquitin can be, for example, a full-length protein. Upon entering the host cell cytosol, ubiquitin expressed from the nucleic acid constructs provided herein can be hydrolytically cleaved at the carboxy terminus from the remainder of the recombinant fusion polypeptide expressed from the nucleic acid construct. This cleavage liberates the amino terminus of the fusion polypeptide, resulting in the production of a peptide in the host cell cytosol.

[0242] The selection, variability, and configuration of antigenic peptides in fusion polypeptides are discussed in detail elsewhere herein, and examples of disease-associated antigenic peptides are discussed in more detail elsewhere herein.

[0243] The recombinant fusion polypeptide can include one or more tags. For example, the recombinant fusion polypeptide can include one or more peptide tags N-terminal and / or C-terminal to one or more antigenic peptides. The tags can be fused directly to the antigenic peptides or linked to the antigenic peptides via a linker (examples of which are disclosed elsewhere herein). Examples of tags include the following: FLAG tag, 2xFLAG tag, 3xFLAG tag, His tag, 6xHis tag, and SIINFEKL tag. An exemplary SIINFEKL tag is set forth in SEQ ID NO: 16 (encoded by any one of the nucleic acids set forth in SEQ ID NOs: 1-15). An exemplary 3xFLAG tag is set forth in SEQ ID NO: 32 (encoded by any one of the nucleic acids set forth in SEQ ID NOs: 17-31). An exemplary variant 3xFLAG tag is set forth in SEQ ID NO: 99. Two or more tags can be used together, such as a 2xFLAG tag and a SIINFEKL tag, a 3xFLAG tag and a SIINFEKL tag, or a 6xHis tag and a SIINFEKL tag. When two or more tags are used, they can be located anywhere within the recombinant fusion polypeptide and in any order. For example, two tags can be located at the C-terminus of the recombinant fusion polypeptide, two tags can be located at the N-terminus of the recombinant fusion polypeptide, two tags can be located internally within the recombinant fusion polypeptide, one tag at the C-terminus and one tag at the N-terminus of the recombinant fusion polypeptide, one tag at the C-terminus and one internal tag, or one tag at the N-terminus and one internal tag within the recombinant fusion polypeptide. Other tags include chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), and poly(NANP). Certain recombinant fusion polypeptides contain a C-terminal SIINFEKL tag.Such tags may allow for easy detection of the recombinant fusion protein, confirmation of the secretion of the recombinant fusion protein, or tracking of the immunogenicity of the secreted fusion polypeptide by tracking the immune response to these "tag" sequence peptides. Such immune responses can be monitored using a number of reagents, including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags.

[0244] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains with expression of such antigenic peptides can be used, for example, in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising recombinant Listeria strains and adjuvants. Expression of one or more antigenic peptides as fusion polypeptides with non-hemolytic truncated LLO, ActA, or PEST-like sequences in host cell systems of Listeria strains and in host cell systems other than Listeria can result in enhanced immunogenicity of the antigenic peptides.

[0245] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. It can comprise or consist of DNA or RNA, and can be single-stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or within a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or two or more open reading frames (e.g., an open reading frame encoding a recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can contain two or more open reading frames linked by Shine-Dalgarno ribosome binding site nucleic acid sequences between each open reading frame. For example, the nucleic acid can contain two to four open reading frames linked by Shine-Dalgarno ribosome binding site nucleic acid sequences between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure the termination of protein synthesis. A. Antigenic Peptides

[0246] Disease-related peptides include peptides from proteins expressed during a particular disease. For example, such peptides may be from proteins expressed in diseased tissue but not in corresponding normal tissue, or from proteins expressed at abnormally high levels in diseased tissue. As used herein, the term "disease" is generally intended to be synonymous with the terms "disorder" and "condition" (as in medical conditions), all of which are used interchangeably to refer to an abnormal condition in the human or animal body or one of its parts that impairs normal function, typically manifests as characteristic signs and symptoms, and causes a shortened lifespan or reduced quality of life for the human or animal. Examples of disease-related antigenic peptides include human papillomavirus (HPV) E7 or E6, prostate-specific antigen (PSA), chimeric Her2 antigen, and Her2 / neu chimeric antigen. Another example of a disease-related antigenic peptide is the WT1 antigenic peptide. The human papillomavirus may be HPV16 or HPV18. The antigenic peptides are HPV16 E6, HPV16 E7, HPV18, and HPV19, which are operably linked in tandem. It may also include an E6, HPV18 E7 antigen, or an HPV16 antigenic peptide operably linked in tandem with an HPV antigenic peptide.

[0247] The fusion polypeptide may contain a single antigenic peptide or may contain two or more antigenic peptides, each of which may be of any length sufficient to induce an immune response, and each of which may be the same length or may have different lengths. For example, the antigenic peptides disclosed herein can be 5 to 100, 15 to 50, or 21 to 27 amino acids in length, or can be 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 11 to 21, 15 It can be 21, 21-31, 31-41, 41-51, 51-61, 61-71, 71-81, 81-91, 91-101, 101-121, 121-141, 141-161, 161-181, 181-201, 8-27, 10-30, 10-40, 15-30, 15-40, 15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11, or 11-16 amino acids. For example, antigenic peptides can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. Some specific examples of antigenic peptides are 21 or 27 amino acids in length. Other antigenic peptides can be full-length proteins or fragments thereof.

[0248] As an example, antigenic peptides can contain neoepitopes. These neoepitopes can be, for example, patient-specific (i.e., subject-specific) cancer mutations. Neoepitopes-containing antigenic peptides can be generated in a process for creating personalized immunotherapy, which involves comparing nucleic acids extracted from a cancer sample from a subject with nucleic acids extracted from a normal or healthy reference sample to identify somatic mutations or sequence differences present in the cancer sample compared to the normal or healthy sample. For example, these mutations or sequence differences can be somatic nonsynonymous missense mutations or somatic frameshift mutations and can encode expressed amino acid sequences. Peptides expressing such somatic mutations or sequence differences can be referred to as "neoepitopes." Cancer-specific neoepitopes can also refer to epitopes found in cancer samples but not present in reference samples (e.g., normal, noncancerous, or germline cells or tissues). This includes, for example, situations where a corresponding epitope is found in normal non-cancerous or germline cells, but one or more mutations in cancer cells alter the sequence of the epitope, resulting in a neoepitope, which may include the mutated epitope or may include non-mutated sequence on one or both sides of the mutation.

[0249] As another example, antigenic peptides can contain recurrent cancer mutations. Each antigenic peptide can contain a single recurrent cancer mutation, or two or more recurrent cancer mutations (e.g., two recurrent cancer mutations). For example, an antigenic peptide can contain more than one recurrent cancer mutation (e.g., two or three recurrent cancer mutations) because the mutated residues in the cancer-associated protein are close to each other. The recurrent cancer mutations can be any type of mutation (e.g., somatic missense mutations or frameshift mutations). For example, the recombinant fusion polypeptides disclosed herein can contain a PEST-containing peptide fused to two or more antigenic peptides (i.e., in tandem, e.g., PEST-peptide 1-peptide 2), or two or more antigenic peptides not fused to a PEST-containing peptide, each of which contains a single recurrent cancer mutation (i.e., a single recurrent change in the amino acid sequence of a protein or a sequence encoded by a single, different, non-synonymous, recurrent cancer mutation within a gene), and at least two of the antigenic peptides contain different recurrent cancer mutations and are fragments of the same cancer-associated protein. Alternatively, each of the antigenic peptides may contain a different recurrent cancer mutation from a different cancer-associated protein. Alternatively, a combination of distinct fusion polypeptides can be used, in which each antigenic peptide is fused (or not fused) to its own PEST-containing peptide (e.g., PEST1-peptide1; PEST2-peptide2). Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein. Non-contiguous fragments are fragments that do not occur sequentially in the protein sequence (e.g., a first fragment consisting of residues 10-30 and a second fragment consisting of residues 100-120, or a first fragment consisting of residues 10-30 and a second fragment consisting of residues 20-40). Optionally, each of the antigenic peptides contains a different recurrent cancer mutation from a single type of cancer.

[0250] Recurrent cancer mutations may be from cancer-associated proteins. The term "cancer-associated protein" includes proteins with mutations that occur in multiple types of cancer, proteins with mutations that occur in multiple subjects with a particular type of cancer, or proteins with mutations that correlate with the development or progression of one or more types of cancer. For example, a cancer-associated protein may be an oncogenic protein (i.e., a protein with activity that may contribute to cancer progression, e.g., a protein that regulates cell proliferation) or a tumor suppressor protein (i.e., a protein that generally acts to reduce the likelihood of cancer formation, e.g., by negatively regulating the cell cycle or promoting apoptosis). In some embodiments, a cancer-associated protein has a "mutational hotspot." A mutational hotspot is an amino acid position within a protein-coding gene that is mutated more frequently (e.g., by somatic substitutions, e.g., translocations, amplifications, and deletions, rather than somatic abnormalities) than would be expected in the absence of selection. Such hotspot mutations may occur across multiple types of cancer and / or be shared among multiple patients. Mutational hotspots represent selective pressure across a population of tumor samples. Tumor genomes contain recurrent cancer mutations that "drive" tumor development by acting on genes (i.e., tumor driver genes) that, when altered, confer a selective growth advantage to tumor cells. Such tumor driver genes can be identified, for example, by identifying genes (i.e., repeats) that are mutated more frequently than expected from background mutation rates; by identifying genes that exhibit other positive selection signals across tumor samples (e.g., a high rate of non-silent mutations compared to silent mutations, or a bias toward the accumulation of functional mutations); by exploiting the tendency to persist mutations in certain regions of a protein sequence, based on the finding that inactivating mutations are distributed along the protein sequence, while gain-of-function mutations tend to occur specifically at certain residues or domains; or by exploiting the overrepresentation of mutations in specific functional residues, such as phosphorylation sites.Many of these mutations often occur in functional regions (e.g., kinase domains or binding domains) of biologically active proteins or block active sites (e.g., phosphorylation sites), resulting in loss-of-function or gain-of-function mutations, or they may occur in a manner that does not sufficiently disrupt the three-dimensional structure and / or charge balance of the protein to interfere with normal function. Genomic analysis of a large number of tumors reveals that mutations often occur at a limited number of amino acid positions. Therefore, the majority of common mutations can be represented by a relatively small number of potential tumor-associated antigens or T-cell epitopes.

[0251] A "recurrent cancer mutation" is a change in the amino acid sequence of a protein that occurs in multiple types of cancer and / or in multiple subjects with a particular type of cancer. Such cancer-associated mutations can result in tumor-associated antigens that are not normally present in corresponding healthy tissue.

[0252] Tumor driver genes and cancer-associated proteins with common mutations that occur across multiple cancers or among multiple cancer patients are known, and sequence data exist across multiple tumor samples and multiple tumor types. See, e.g., Chang et al. (2016) See Nat Biotechnol 34(2):155-163; Tamborero et al. (2013) Sci Rep 3:2650, each of which is incorporated herein by reference in its entirety.

[0253] As another example, the antigenic peptide can be a heteroclitic antigenic peptide. For example, the heteroclitic antigenic peptide can be a fragment of a cancer-associated protein (i.e., a consecutive sequence of amino acids from a cancer-associated protein) containing a heteroclitic mutation. The heteroclitic antigenic peptide can contain a single heteroclitic mutation, or can contain two or more heteroclitic mutations (e.g., two heteroclitic mutations). The term "heteroclitic" refers to a peptide that elicits an immune response that recognizes the native peptide from which the heteroclitic peptide was derived (e.g., a peptide that does not contain anchor residue mutations).

[0254] Some recombinant fusion polypeptides disclosed herein may contain any combination of antigenic peptides comprising recurrent cancer mutations, antigenic peptides comprising heteroclitic mutations (e.g., from cancer-associated proteins), and antigenic peptides expressed from minigene constructs (e.g., from cancer-associated proteins) (i.e., antigenic peptides such as heteroclitic antigenic peptides fused to ubiquitin). For example, such a recombinant fusion polypeptide may contain a PEST-containing peptide fused to two or more antigenic peptides, at least one of which is from a cancer-associated protein and comprises a recurrent cancer mutation, and at least one of which is from a cancer-associated protein and comprises a heteroclitic mutation. Optionally, the PEST-containing peptide comprises a bacterial secretion signal sequence, and the fusion polypeptide further comprises a ubiquitin protein fused to the carboxy-terminal antigenic peptide, wherein the PEST-containing peptide, the two or more antigenic peptides, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from the amino terminus to the carboxy terminus of the fusion protein.

[0255] Each antigenic peptide may also be hydrophilic or have a score at or below a certain hydropathy threshold that may be predictive of secretion potential in Listeria monocytogenes or another bacterium of interest. For example, antigenic peptides may be scored using a Kyte and Doolittle hydropathy index 21-amino acid window, and all with scores above the cutoff (approximately 1.6) may be eliminated because they are unlikely to be secreted by Listeria monocytogenes. Similarly, antigenic peptide or fusion polypeptide combinations may be hydrophilic or have a score at or below a certain hydropathy threshold that may be predictive of secretion potential in Listeria monocytogenes or another bacterium of interest.

[0256] Antigenic peptides can be linked to each other in any manner. For example, antigenic peptides can be directly fused to each other without any intervening sequence. Alternatively, antigenic peptides can be indirectly linked to each other via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be directly fused to each other, and other pairs of antigenic peptides can be indirectly linked to each other via one or more linkers. The same linker can be used between each pair of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. In addition, one linker can be used between pairs of adjacent antigenic peptides, or multiple linkers can be used between pairs of adjacent antigenic peptides.

[0257] Any suitable sequence can be used for the peptide linker. For example, the linker sequence can be, for example, 1 to about 50 amino acids in length. Some linkers can be hydrophilic. Linkers can serve various purposes. For example, linkers can increase bacterial secretion, facilitate antigen processing, increase the flexibility of the fusion polypeptide, increase the rigidity of the fusion polypeptide, or any other purpose. In some cases, to minimize repetition, different amino acid linker sequences are distributed between antigen peptides, or different nucleic acids encoding the same amino acid linker sequence are distributed between antigen peptides (e.g., SEQ ID NOS: 84-94). This can also help reduce secondary structure, thereby enabling efficient transcription, translation, secretion, maintenance, or stabilization of nucleic acids (e.g., plasmids) encoding fusion polypeptides within the Lm recombinant vector strain population. Other suitable peptide linker sequences can be selected based on, for example, one or more of the following factors: (1) the ability to adopt a flexible extended conformation; (2) the inability to adopt a secondary structure that can interact with a functional epitope on the antigen peptide; and (3) the lack of hydrophobic or charged residues that may react with the functional epitope. For example, the peptide linker sequence can contain Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be used to serve as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA 83:8258-8262; US 4,935,233; and US 4,751,180, each of which is incorporated herein by reference in its entirety for all purposes.Specific examples of linkers include those in Table 2 (each of which may be used alone as a linker, in a linker that includes repeats of the sequence, or in a linker that further includes one or more of the other sequences in the table), although others are envisioned (see, e.g., Reddy Chichili et al. (2013) Protein Science 22:153-167, which is incorporated by reference in its entirety for all purposes). Unless specified, "n" represents the undetermined number of repeats in the listed linker.

[0258] [Table 2] B. PEST-containing peptides

[0259] The recombinant fusion proteins disclosed herein contain a PEST-containing peptide. The PEST-containing peptide may be at the amino-terminus (N-terminus) of the fusion polypeptide (i.e., the N-terminus of the antigenic peptide), at the carboxy-terminus (C-terminus) of the fusion polypeptide (i.e., the C-terminus of the antigenic peptide), or embedded within the antigenic peptide. In some recombinant Listeria strains and methods, the PEST-containing peptide is not part of the fusion polypeptide but is separate from the fusion polypeptide. Fusing an antigenic peptide, such as an LLO peptide, to a PEST-like sequence can enhance the immunogenicity of the antigenic peptide and increase cell-mediated anti-tumor immune responses (i.e., increase cell-mediated anti-tumor immunity). See, e.g., Singh et al. (2005) J Immunol 175(6):3663-3673, incorporated herein by reference in its entirety for all purposes.

[0260] PEST-containing peptides are those that contain PEST sequence or PEST-like sequence. PEST sequence in eukaryotic proteins has been identified for a long time. For example, proteins containing amino acid sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST), which are generally but not always adjacent to clusters containing several positively charged amino acids, have rapid intracellular half-life (Rogers et al. (1986) Science 234:364-369, the entire contents of which are incorporated herein by reference for all purposes). Furthermore, these sequences have been reported to target proteins to the ubiquitin-proteasome pathway for degradation (Rechsteiner et al. (1986) Science 234:364-369, the entire contents of which are incorporated herein by reference for all purposes). and Rogers (1996) Trends Biochem. Sci. 21:267-271). This pathway is also used by eukaryotic cells to generate immunogenic peptides that bind to MHC class I, and it has been hypothesized that PEST sequences are abundant in eukaryotic proteins that give rise to immunogenic peptides (Realini et al. (1994) FEBS Lett. 348:109-113, incorporated herein by reference in its entirety for all purposes). Prokaryotic proteins do not typically contain PEST sequences because they do not possess this enzymatic pathway. However, a PEST-like sequence rich in the amino acids proline (P), glutamic acid (E), serine (S), and threonine (T) has been reported at the amino terminus of LLO and is reported to be essential for L. monocytogenes virulence (Decatur and Portnoy (2000) Science 290:992-995, incorporated herein by reference in its entirety for all purposes). The presence of this PEST-like sequence in LLO targets the protein for degradation by the host cell's proteolytic machinery, such that after LLO has performed its function and facilitated the escape of L. monocytogenes from the phagosome or phagolysosomal vacuole, the protein is destroyed before it can cause cell damage.

[0261] The identification of PEST and PEST-like sequences is well known and is described, for example, in Rogers et al. (1986) Science 234(4774):364-378 and Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21:267-271, each of which is incorporated herein by reference in its entirety for all purposes. PEST or PEST-like sequences can be identified using a PEST discovery program. For example, a PEST-like sequence can be a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. Optionally, a PEST-like sequence can be flanked by one or more clusters containing several positively charged amino acids. For example, a PEST-like sequence can be defined as a hydrophilic stretch of at least 12 amino acids in length with a high local concentration of proline (P), aspartate (D), glutamate (E), serine (S), and / or threonine (T) residues. In some cases, PEST-like sequences do not contain positively charged amino acids, i.e., arginine (R), histidine (H), and lysine (K). Some PEST-like sequences may contain one or more internal phosphorylation sites, and phosphorylation at these sites precedes proteolysis.

[0262] In one example, PEST-like sequences fit the algorithm disclosed in Rogers et al. In another example, PEST-like sequences fit the algorithm disclosed in Rechsteiner and Rogers. PEST-like sequences can also be identified by first scanning for the positively charged amino acids R, H, and K within a specified protein sequence. All amino acids flanked by positive charges are counted, and only motifs containing a number of amino acids equal to or greater than the window size parameter are further considered. Optionally, PEST-like sequences must contain at least one P, at least one D or E, and at least one S or T.

[0263] The quality of PEST motifs can be improved by scoring parameters based on the local enrichment of key amino acids and the hydrophobicity of the motif. Enrichments of D, E, P, S, and T are expressed as mass percent (w / w) and corrected for 1 equivalent of D or E, 1 equivalent of P, and 1 equivalent of S or T. Hydrophobicity calculations may also follow essentially the method of Kyte and Doolittle (1982) J. Mol. Biol. 157:105, which is incorporated herein by reference in its entirety for all purposes. For simplified calculations, the Kyte·Doolittle hydropathic index, which originally ranges from −4.5 for arginine to +4.5 for isoleucine, was converted to a positive integer using the following linear transformation, which yielded values ​​ranging from 0 for arginine to 90 for isoleucine: hydropathic index = 10 × Kyte·Doolittle hydropathic index + 45.

[0264] The hydrophobicity of a potential PEST motif can also be calculated as the sum of the products of the mole percent and hydrophobicity index for each amino acid species. The desired PEST score is obtained as a combination of the local enrichment and hydrophobicity terms, as represented by the following equation: PEST score = 0.55 × DEPST - 0.5 × hydrophobicity index.

[0265] Thus, a PEST-containing peptide can refer to a peptide having a score of at least +5 using the above algorithm. Alternatively, a PEST-containing peptide can refer to a peptide having a score of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 35, at least 38, at least 40, or at least 45.

[0266] Any other known and available method or algorithm can also be used to identify PEST-like sequences. For example, see CaSPredictor (Garay-Malpartida et al. (2005) Bioinformatics 21 Suppl 1:i169-76, the entire contents of which are incorporated herein by reference for all purposes). Another method that can be used is as follows: the PEST index is calculated for each stretch of appropriate length (e.g., a 30-35 amino acid stretch) by assigning a value of 1 to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gln. The coefficient value (CV) for each PEST residue is 1, and the CV for each other AA (non-PEST) is zero.

[0267] Examples of PEST-like amino acid sequences are set forth in SEQ ID NOS: 43-51. One example of a PEST-like sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 43). Another example of a PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID NO: 44). However, any PEST or PEST-like amino acid sequence can be used. PEST sequence peptides are known and are described, for example, in US Pat. No. 7,635,479, US Pat. No. 7,665,238, and US 2014 / 0186387, each of which is incorporated herein by reference in its entirety for all purposes.

[0268] The PEST-like sequence can be from a Listeria species, e.g., Listeria monocytogenes. For example, the Listeria monocytogenes ActA protein contains at least four such sequences (SEQ ID NOS: 45-48), any of which are suitable for use in the compositions and methods disclosed herein. Other similar PEST-like sequences include SEQ ID NOS: 52-54. Streptolysin O proteins from Streptococcus sp. also contain PEST sequences. For example, Streptococcus pyogenes streptolysin O contains the PEST sequence KQNTASTETTTTNEQPK (SEQ ID NOS: 49) at amino acids 35-51, and Streptococcus equisimilis streptolysin O contains the PEST-like sequence KQNTANTETTTTNEQPK (SEQ ID NOS: 50) at amino acids 38-54. Another example of a PEST-like sequence is RSEVTISPAETPESPPATP (e.g., SEQ ID NOS: 51) from Listeria seeligeri cytolysin, encoded by the lso gene.

[0269] Alternatively, PEST-like sequences may be derived from other prokaryotes, including, for example, other Listeria species, for which PEST-like amino acid sequences are predicted. (1) Listeriolysin O (LLO)

[0270] One example of a PEST-containing peptide that can be utilized in the compositions and methods disclosed herein is the listeriolysin O (LLO) peptide. An example of an LLO protein is the protein designated by GenBank Accession No. P13128 (SEQ ID NO:55; the nucleic acid sequence is set forth in GenBank Accession No. X15127). SEQ ID NO:55 is a proprotein that includes a signal sequence. The first 25 amino acids of this proprotein are the signal sequence, which is cleaved from LLO upon secretion by bacteria, resulting in the 504 amino acid full-length active LLO protein without the signal sequence. The LLO peptides disclosed herein may include signal sequences or peptides without a signal sequence. Exemplary LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO:55, or homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO:55. Any sequence encoding a fragment of an LLO protein, or a homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant, or fragment of an analog of an LLO protein can be used. A homologous LLO protein can have greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference LLO protein.

[0271] Another example of an LLO protein is set forth in SEQ ID NO: 56. LLO proteins that can be used may comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 56, or homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 56.

[0272] Another example of an LLO protein is the LLO protein from Listeria monocytogenes strain 10403S, as set forth in GenBank Accession Nos. ZP_01942330 or EBA21833, or as encoded by the nucleic acid sequence set forth in GenBank Accession Nos. NZ_AARZ01000015 or AARZ01000015.1. Another example of an LLO protein is the LLO protein from Listeria monocytogenes strain 4b F2365 (see, e.g., GenBank Accession No. YP_012823), the LLO protein from the EGD-e strain (see, e.g., GenBank Accession No. NP_463733), or an LLO protein from any other strain of Listeria monocytogenes. Yet another example of an LLO protein is the LLO protein from Flavobacteriales bacterium HTCC2170 (see, e.g., GenBank accession number ZP_01106747 or EAR01433, or encoded by GenBank accession number NZ_AAOC01000003). LLO proteins that can be used can comprise, consist essentially of, or consist of the above-mentioned LLO proteins, or any of the above-mentioned LLO protein homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms.

[0273] Homologous proteins of LLO, or their homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms, can also be used. One such example is alveologen, which can be found, for example, in Paenibacillus alvei (see, e.g., GenBank Accession Nos. P23564 or AAA22224, or encoded by GenBank Accession No. M62709). Other such homologous proteins are known.

[0274] The LLO peptide can be a full-length LLO protein, a truncated LLO protein, or an LLO fragment. Similarly, the LLO peptide can retain one or more functionalities of the native LLO protein or can lack one or more functionalities of the native LLO protein. For example, the retained LLO functionality can enable escape from a phagosome or phagolysosome by bacteria (e.g., Listeria) or enhance the immunogenicity of the peptide fused thereto. The retained functionality can also be a hemolytic or antigenic function. Alternatively, the LLO peptide can be a non-hemolytic LLO. Other functions of LLO are known, and methods and assays for assessing LLO functionality are also known.

[0275] An LLO fragment may be or contain a PEST-like sequence. An LLO fragment may contain one or more of an internal deletion, a truncation from the C-terminus, and a truncation from the N-terminus. In some cases, an LLO fragment may contain more than one internal deletion. Other LLO peptides may be full-length LLO proteins with one or more mutations.

[0276] Some LLO proteins or fragments have reduced hemolytic activity compared to wild-type LLO, or are non-hemolytic fragments. For example, LLO proteins can be made non-hemolytic by deleting or mutating the activation domain at the carboxy terminus, by deleting or mutating cysteine ​​484, or by deleting or mutating at another position.

[0277] Other LLO proteins are made non-hemolytic by deleting or mutating the cholesterol binding domain (CBD), as described in US8,771,702, the entirety of which is incorporated herein by reference for all purposes.Mutation can include, for example, substitution or deletion.The entire CBD can be mutated, a portion of the CBD can be mutated, or specific residues within the CBD can be mutated.For example, the LLO protein can include mutations of one or more of residues C484, W491 and W492 of SEQ ID NO:55 (e.g., C484, W491, W492, C484 and W491, C484 and W492, W491 and W492, or all three residues), or mutations of residues that match when optimally aligned with SEQ ID NO:55 (e.g., matching cysteine ​​or tryptophan residues). As an example, a mutant LLO protein can be created in which residues C484, W491, and W492 of LLO are substituted with alanine residues, resulting in a substantially reduced hemolytic activity compared to wild-type LLO. A mutant LLO protein with the C484A, W491A, and W492A mutations is referred to as "mutLLO."

[0278] As another example, a mutant LLO protein can be created with an internal deletion that includes the cholesterol-binding domain. The sequence of the cholesterol-binding domain of SEQ ID NO: 55 is set forth in SEQ ID NO: 74. For example, the internal deletion can be a 1-11 amino acid deletion, or a 11-50 amino acid deletion, or longer. Similarly, the mutated region may be 1-11 amino acids, 11-50 amino acids, or longer (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11 ~35, 11~40, 11~50, 11~60, 11~70, 11~80, 11~90, 11~100, 11~150, 15~20, 15~25, 15~30, 15~35, 15~40, 15~50, 15~60, 15~70, 15~80, 15~90, 15~100, 15~150 , 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). For example, a mutated region consisting of residues 470-500, 470-510, or 480-500 of SEQ ID NO: 55 would result in a deleted sequence containing the CBD (residues 483-493 of SEQ ID NO: 55). However, the mutated region can also be a fragment of or overlap with a portion of the CBD. For example, the mutated region can consist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or 486-510 of SEQ ID NO: 55. For example, a fragment of the CBD (residues 484-492) can be replaced with a heterologous sequence, resulting in a substantially reduced hemolytic activity compared to wild-type LLO.For example, the CBD (ECTGLAWEWWR; SEQ ID NO: 74) can be replaced with a CTL epitope from antigen NY-ESO-1 (ESLLMWITQCR; SEQ ID NO: 75), which contains HLA-A2-restricted epitopes 157-165 from NY-EOS-1. The resulting LLO is referred to as "ctLLO."

[0279] In some mutated LLO proteins, the mutated region may be replaced by a heterologous sequence. For example, the mutated region may contain an equal number of heterologous amino acids, fewer heterologous amino acids, or a greater number of amino acids (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, The amino acids may be replaced by 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids. Other mutated LLO proteins have one or more point mutations (e.g., point mutations of one, two, three, or more residues). The mutated residues may be contiguous or non-contiguous.

[0280] In one example embodiment, the LLO peptide may have a deletion in the signal sequence and a mutation or substitution in the CBD.

[0281] Some LLO peptides are N-terminal LLO fragments (i.e., LLO proteins with C-terminal deletions). Some LLO peptides are at least 494, 489, 492, 493, 500, 505, 510, 515, 520, or 525 amino acids in length, or 492 to 528 amino acids in length. For example, an LLO fragment can consist of approximately the first 440 or 441 amino acids of an LLO protein (e.g., the first 441 amino acids of SEQ ID NO: 55 or 56, or a fragment that matches when optimally aligned with SEQ ID NO: 55 or 56 of another LLO protein). Other N-terminal LLO fragments can consist of the first 420 amino acids of an LLO protein (e.g., the first 420 amino acids of SEQ ID NO: 55 or 56, or a fragment that matches when optimally aligned with SEQ ID NO: 55 or 56 of another LLO protein). Other N-terminal fragments can consist of about amino acids 20-442 of the LLO protein (e.g., amino acids 20-442 of SEQ ID NO: 55 or 56, or a fragment that matches when optimally aligned with SEQ ID NO: 55 or 56 of another LLO protein). Other N-terminal LLO fragments include any ΔLLO without the activation domain containing cysteine ​​484, particularly without cysteine ​​484. For example, an N-terminal LLO fragment can correspond to the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of an LLO protein (e.g., the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of SEQ ID NO: 55 or 56, or a fragment that matches when optimally aligned with SEQ ID NO: 55 or 56 of another LLO protein). In some embodiments, the fragment comprises one or more PEST-like sequences. LLO fragments and truncated LLO proteins can contain residues of a homologous LLO protein corresponding to any one of the above specific amino acid ranges. The residue numbers need not exactly correspond to those listed above (eg, if the homologous LLO protein has insertions or deletions compared to the particular LLO proteins disclosed herein).Examples of N-terminal LLO fragments include SEQ ID NOs: 57, 58, and 59. LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NOs: 57, 58, or 59, or homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NOs: 57, 58, or 59. Some compositions and methods use the N-terminal LLO fragment set forth in SEQ ID NO: 59. An example of a nucleic acid sequence encoding the N-terminal LLO fragment set forth in SEQ ID NO: 59 is SEQ ID NO: 60. (2) ActA

[0282] Another example of a PEST-containing peptide that can be utilized in the compositions and methods disclosed herein is the ActA peptide. ActA is a surface-binding protein that acts as a scaffold to facilitate the polymerization, assembly, and activation of host actin polymers in infected host cells, allowing Listeria monocytogenes to propel itself through the cytoplasm. Shortly after entering the mammalian cell cytosol, L. monocytogenes induces the polymerization of host actin filaments and uses the forces generated by actin polymerization to move first intracellularly and then from cell to cell. ActA is involved in mediating actin nucleation and actin-based motility. The ActA protein acts as a scaffold to assemble the cellular actin polymerization machinery by providing multiple binding sites for host cytoskeletal components. The N-terminus of ActA binds to monomeric actin and stimulates endogenous actin nucleation activity, acting as a constitutively active nucleation promoter. Both actA and hly genes are members of a 10-kb gene cluster regulated by the transcriptional activator PrfA, with actA being upregulated approximately 226-fold in mammalian cytosol. Any sequence encoding an ActA protein, or a homolog, variant, isoform, analog, or fragment of a homolog, variant, or analog of an ActA protein can be used. A homologous ActA protein can have sequence identity with a reference ActA protein, for example, greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%.

[0283] One example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 61. Another example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 62. The first 29 amino acids of the proprotein corresponding to either of these sequences are signal sequences that are cleaved from the ActA protein when the proprotein is secreted by bacteria. ActA peptides may include a signal sequence (e.g., amino acids 1-29 of SEQ ID NO: 61 or 62) or may include peptides without a signal sequence. Other examples of ActA proteins comprise, consist essentially of, or consist of a homolog, variant, isoform, analog, fragment, fragment of a homolog, fragment of an isoform, or fragment of an analog of SEQ ID NO: 61 or 62.

[0284] Other examples of ActA proteins are the ActA protein from Listeria monocytogenes strain 10403S (GenBank accession number DQ054585), the ActA protein from NICPBP strain 54002 (GenBank accession number EU394959), the ActA protein from strain S3 (GenBank accession number EU394960), the ActA protein from strain NCTC 5348 (GenBank accession number EU394961), the ActA protein from strain NICPBP 54006 (GenBank accession number EU394962), the ActA protein from strain M7 (GenBank accession number EU394963), the ActA protein from strain S19 (GenBank accession number EU394964), or the ActA protein from any other strain of Listeria monocytogenes. The LLO proteins that can be used may comprise, consist essentially of, or consist of the above-mentioned LLO proteins, or any of the homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms of the above-mentioned LLO proteins.

[0285] The ActA peptide can be a full-length ActA protein, a truncated ActA protein, or an ActA fragment (e.g., an N-terminal ActA fragment with the C-terminal portion removed). In some embodiments, the truncated ActA protein contains at least one PEST sequence (e.g., more than one PEST sequence). In addition, the truncated ActA protein may optionally contain an ActA signal peptide. Examples of PEST-like sequences contained in truncated ActA proteins include SEQ ID NOs: 45-48. Some such truncated ActA proteins contain at least two of the PEST-like sequences set forth in SEQ ID NOs: 45-48 or their homologs, at least three of the PEST-like sequences set forth in SEQ ID NOs: 45-48 or their homologs, or all four of the PEST-like sequences set forth in SEQ ID NOs: 45-48 or their homologs. Examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 30-122, about residues 30-229, about residues 30-332, about residues 30-200, or about residues 30-399 of a full-length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about the first 50, 100, 150, 200, 233, 250, 300, 390, 400, or 418 residues of a full-length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 200-300 or 300-400 of a full-length ActA protein sequence (e.g., SEQ ID NO: 62). For example, a truncated ActA consists of the first 390 amino acids of the wild-type ActA protein as described in US Pat. No. 7,655,238, which is incorporated herein by reference in its entirety for all purposes.As another example, the truncated ActA can be ActA-N100 or a modified version thereof (referred to as ActA-N100*), as described in US2014 / 0186387, the entire contents of which are incorporated herein by reference for all purposes, in which the PEST motif is deleted and the non-conservative QDNKR (SEQ ID NO: 73) substitution is contained. Alternatively, the truncated ActA protein can contain residues of a homologous ActA protein corresponding to one of the amino acid ranges described above or any of the ActA peptides disclosed herein. The residue numbers need not exactly match those listed herein (e.g., if the homologous ActA protein has an insertion or deletion compared to the ActA protein used herein, the residue numbers can be adjusted accordingly).

[0286] Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 63, 64, 65, or 66, or a homolog, variant, isoform, analog, or fragment of a variant, isoform, or analog of SEQ ID NO: 63, 64, 65, or 66. SEQ ID NO: 63 is referred to as ActA / PEST1 and consists of amino acids 30-122 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 64 is referred to as ActA / PEST2 or LA229 and consists of amino acids 30-229 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 65 is referred to as ActA / PEST3 and consists of amino acids 30-332 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 66 is referred to as ActA / PEST4 and consists of amino acids 30-399 of the full-length ActA sequence set forth in SEQ ID NO: 62. As a specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 64 can be used.

[0287] Examples of truncated ActA proteins include proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 67, 69, 70, or 72, or a homolog, variant, isoform, analog, fragment of a variant, fragment of an isoform, or fragment of an analog of SEQ ID NO: 67, 69, 70, or 72. As a specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 67 (encoded by the nucleic acid set forth in SEQ ID NO: 68) can be used. As another specific example, a truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 70 (encoded by the nucleic acid set forth in SEQ ID NO: 71) can be used. SEQ ID NO: 71 is the first 1170 nucleotides encoding ActA in Listeria monocytogenes strain 10403S. In some cases, the ActA fragment can be fused to a heterologous signal peptide. For example, SEQ ID NO: 72 shows an ActA fragment fused to the Hly signal peptide. C. Generation of immunotherapeutic constructs encoding recombinant fusion polypeptides

[0288] Also provided herein are methods for producing immunotherapeutic constructs encoding the recombinant fusion polypeptides disclosed herein or compositions comprising said recombinant fusion polypeptides. For example, such methods can include selecting and designing antigenic peptides for inclusion in an immunotherapeutic construct (and, for example, testing the hydropathy of each antigenic peptide and modifying or deselecting an antigenic peptide if it has a score above a selected hydropathy index threshold), designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptides.

[0289] Antigenic peptides can be screened for hydrophobicity or hydrophilicity. Antigenic peptides can be selected, for example, if they are hydrophilic or if they have a score at or below a certain hydropathy threshold, which may predict the secretion potential of a specific bacterium of interest (e.g., Listeria monocytogenes). For example, antigenic peptides can be scored in a 21 amino acid window by Kyte and Doolittle hydropathy index, and all those with scores above the cutoff (approximately 1.6) are excluded. They are likely to be secreted by Listeria monocytogenes. This is because they are less likely to be secreted by S. monocytogenes. See, for example, Kyte-Doolittle (1982) J. Mol. Biol. 157(1):105-132, the entire contents of which are incorporated herein by reference for all purposes. Alternatively, antigenic peptides having a score at about the selection cutoff can be modified (e.g., by changing the length of the antigenic peptide). Other sliding window sizes that can be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or more amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19-21 amino acids, 21-23 amino acids, 23-25 ​​amino acids, or 25-27 amino acids. Other cutoffs that can be used include, for example, the following ranges: 1.2 to 1.4, 1.4 to 1.6, 1.6 to 1.8, 1.8 to 2.0, 2.0 to 2.2, 2.2 to 2.5, 2.5 to 3.0, 3.0 to 3.5, 3.5 to 4.0, or 4.0 to 4.5, or the cutoff can be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. The cut-off may vary, for example, depending on the genus or species of bacteria used to deliver the fusion polypeptide.

[0290] Other suitable hydropathy plots or other suitable measures are described, for example, in Rose et al. al. (1993) Annu Rev Biomol Struct 22:381-415;Biswas et al. (2003) Journal of Chromatography A 1000:637-655;Eisenberg (1984) Ann Rev Biochem 53:595-623;Abraham and Leo (1987) Proteins: Structure, Function and Genetics 2:130-152;Sweet and Eisenberg (1983) Mol Biol 171:479-488;Bull and Breese (1974) Arch Biochem Biophys 161:665-670;Guy (1985) Biophys J 47:61-70;Miyazawa et al. (1985) Macromolecules 18:534-552;Roseman (1988) J Mol Biol 200:513-522;Wolfenden et al. (1981) Biochemistry 20:849-855;Wilson (1981) Biochem J 199:31-41;Cowan and Whittaker (1990) Peptide Research 3:75-80;Aboderin (1971) Int J Biochem 2:537-544;Eisenberg et al. (1984) J Mol Biol 179:125-142;Hopp and Woods (1981) Proc Natl Acad Sci USA 78:3824-3828;Manavalan and Ponnuswamy (1978) Nature 275:673-674;Black and Mould (1991) Anal Biochem 193:72-82;Fauchere and Pliska (1983) Eur J Med Chem 18:369-375;Janin (1979) Nature 277:491-492;Rao and Argos (1986) Biochim Biophys Acta 869:197-214; Tanford (1962) Am Chem Soc 84:4240-4274; Welling et al. (1985) FEBS Lett 188:215-218; Parker et al. (1986) Biochemistry 25:5425-5431; and Cowan and Whittaker (1990) Peptide Research 3:75-80, each of which is incorporated herein by reference in its entirety for all purposes.

[0291] Optionally, antigenic peptides can be scored for their ability to bind to target human leukocyte antigen (HLA) types (e.g., by using the Immune Epitope Database (IED), available at www.iedb.org, including netMHCpan, ANN, SMMPMBEC.SMM, CombLib_Sidney2008, PickPocket, and netMHCcons) and ranked by the highest MHC binding score from each antigenic peptide. Other sources include TEpredict (tepredict.sourceforge.net / help.html) or other available MHC binding metrics. Cutoffs may vary for different expression vectors, e.g., Salmonella.

[0292] If necessary, the antigenic peptides can be screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing helper T epitopes, etc.) to deselect the antigenic peptide or avoid its immunosuppressive effects.

[0293] Optionally, antigenic peptides can be screened using predictive algorithms for epitope immunogenicity. However, these algorithms have an accuracy rate of up to 20% in predicting which peptides will elicit a T cell response. Alternatively, no screening / predictive algorithms are used. Alternatively, antigenic peptides can be screened for immunogenicity. For example, the screening can include contacting one or more T cells with the antigenic peptide and analyzing for an immunogenic T cell response, where an immunogenic T cell response identifies the peptide as an immunogenic peptide. The screening can also include using an immunogenicity assay to measure the secretion of at least one of CD25, CD44, or CD69, or a cytokine selected from the group consisting of IFN-γ, TNF-α, IL-1, and IL-2, by contacting one or more T cells with the peptide, where an increase in secretion identifies the peptide as containing one or more T cell epitopes.

[0294] Selected antigenic peptides can be arranged in one or more candidate orders for potential fusion polypeptides. If antigenic peptides are available in addition to being able to fit onto a single plasmid, different antigenic peptides can be prioritized as necessary / desired and / or distributed among different fusion polypeptides (e.g., for incorporation into different recombinant Listeria strains). Priority can be determined by factors such as the relative size, transcriptional preference, and / or overall hydrophobicity of the translated polypeptide. As disclosed in more detail elsewhere herein, antigenic peptides can be arranged so that they are directly linked between any number of antigenic peptide pairs without a linker or any combination of linkers. The number of linear antigenic peptides to be included can be determined based on considerations of the number of constructs required relative to the amount of mutation, the efficiency of translation and secretion of multiple epitopes from a single plasmid, and the MOI required for each bacterium or Lm containing the plasmid.

[0295] The combination of antigenic peptides or the entire fusion polypeptide (i.e., including the antigenic peptide, PEST-containing peptide, and optional tag) is scored for hydrophobicity in the same way. For example, the entire fused antigenic peptide or the entire fusion polypeptide can be scored for hydrophobicity using the Kyte and Doolittle hydrophobicity index in a sliding 21-amino acid window. If any region has a score above the cutoff (e.g., approximately 1.6), the antigenic peptides can be rearranged or shuffled within the fusion polypeptide until an acceptable order of antigenic peptides (i.e., one in which no region has a score above the cutoff) is found. Alternatively, any problematic antigenic peptides can be removed or redesigned to be of a different size. Alternatively or additionally, one or more linkers between antigenic peptides can be added or modified to change hydrophobicity, as disclosed elsewhere herein. As with the hydrophobicity test for individual antigenic peptides, other window sizes can be used, or other cutoffs can be used (e.g., depending on the genus or species of bacteria used to deliver the fusion polypeptide). Additionally, other suitable hydropathy plots or other appropriate measures could be used.

[0296] If desired, the antigenic peptide combination or the entire fusion polypeptide can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing helper T epitopes, etc.) to deselect the antigenic peptide or avoid its immunosuppressive effects.

[0297] Nucleic acids encoding candidate antigen peptide combinations or fusion polypeptides can then be designed and optimized. For example, sequences can be optimized to increase translation levels, expression duration, secretion levels, transcription levels, and any combination thereof. For example, increases can be 2-fold to 1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 20-fold, 2-fold to 10-fold, or 3-fold to 5-fold compared to a control, non-optimized sequence.

[0298] For example, fusion polypeptides or nucleic acids encoding fusion polypeptides can be optimized to reduce the level of secondary structures that may form in the oligonucleotide sequence, or alternatively, to prevent the binding of any enzymes that may modify the sequence. Expression in bacterial cells can be hindered by, for example, transcriptional silencing, short mRNA half-life, secondary structure formation, binding sites for oligonucleotide-binding molecules such as repressors and inhibitors, and the availability of rare tRNA pools. The causes of many problems with bacterial expression can be found within the original sequence. RNA optimization can include modifying cis-acting elements, adapting its GC content, correcting codon bias for the non-limiting tRNA pool of bacterial cells, and avoiding internal homologous regions. Therefore, optimizing a sequence can involve, for example, adjusting regions with very high (>80%) or very low (<30%) GC content. Optimizing the sequence may involve, for example, avoiding one or more of the following cis-acting motifs: internal TATA boxes, Chi sites, and ribosome entry sites; AT-rich or GC-rich sequence stretches; repetitive sequences and RNA secondary structures; (cryptic) splice donor and acceptor sites; branch points; or combinations thereof. Optimizing expression may also involve adding sequence elements adjacent to the gene and / or elsewhere within the plasmid.

[0299] Optimizing a sequence can also involve, for example, adapting codon usage to the codon bias of a host gene (e.g., a Listeria monocytogenes gene). For example, codons that may be used in Listeria monocytogenes include: A=GCA, G=GGT, L=TTA, Q=CAA, V=GTT, C=TGT, H=CAT, M=ATG, R=CGT, W=TGG, D=GAT, I=ATT, N=AAC, S=TCT, Y=TAT, E=GAA, K=AAA, P=CCA, T=ACA, F=TTC, and STOP=TAA.

[0300] The nucleic acid encoding the fusion polypeptide can be produced and introduced into a delivery vehicle, for example, a bacterial strain or a Listeria strain.Other delivery vehicles, such as vaccinia virus or virus-like particles, can be suitable for DNA immunotherapy or peptide immunotherapy.After the plasmid encoding the fusion polypeptide is produced and introduced into a bacterial strain or a Listeria strain, the bacteria or Listeria strain can be cultured and characterized to confirm the expression and secretion of the fusion polypeptide containing the antigenic peptide. V. Immunogenic Compositions, Pharmaceutical Compositions and Vaccines

[0301] Also provided are immunogenic compositions, pharmaceutical compositions, or vaccines comprising lyophilized recombinant bacteria or Listeria strains as disclosed herein, where the lyophilized recombinant bacteria or Listeria strains are optionally reconstituted by dissolving in a volume of solvent. Immunogenic compositions comprising Listeria strains may be inherently immunogenic because they comprise Listeria strains, and / or the compositions may further comprise an adjuvant. Other immunogenic compositions include DNA immunotherapy or peptide immunotherapy compositions.

[0302] The term "immunogenic composition" refers to any composition containing an antigen that elicits an immune response in a subject against the antigen upon exposure to the composition. The immune response elicited by an immunogenic composition can be directed against a specific antigen or against a specific epitope on the antigen.

[0303] An immunogenic composition may contain a single lyophilized or reconstituted recombinant bacterium or Listeria strain as disclosed herein, or may contain multiple different lyophilized or reconstituted recombinant bacterium or Listeria strains as disclosed herein. A bacterium or Listeria strain containing a first recombinant fusion polypeptide is different from a bacterium or Listeria strain containing a second recombinant fusion polypeptide, for example, if the first recombinant fusion polypeptide contains an antigenic peptide that the second recombinant fusion polypeptide does not contain. Two recombinant fusion polypeptides may contain some of the same antigenic peptides and still be considered different. Such different lyophilized or reconstituted recombinant bacterium or Listeria strains can be administered to a subject simultaneously or sequentially. Sequential administration is particularly useful when the drug substances comprising the lyophilized or reconstituted recombinant Listeria strains (or recombinant fusion polypeptides or nucleic acids) disclosed herein are in different dosage forms and / or are administered on different dosing schedules (e.g., one composition from the mixture is administered at least daily and another is administered less frequently, e.g., once a week, once every two weeks, or once every three weeks). Multiple lyophilized or reconstituted recombinant bacteria or Listeria strains can each comprise a different set of antigenic peptides. Alternatively, two or more lyophilized or reconstituted recombinant bacteria or Listeria strains can comprise the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[0304] The immunogenic composition may further comprise an adjuvant (e.g., two or more adjuvants), a cytokine, a chemokine, or a combination thereof. Optionally, the immunogenic composition may further comprise antigen-presenting cells (APCs), which may be autologous or allogeneic to the subject.

[0305] The term adjuvant includes compounds or mixtures that enhance the immune response to an antigen. For example, an adjuvant can be a non-specific stimulator of the immune response, or a substance that allows the formation of a depot in a subject, which, when combined with the immunogenic compositions disclosed herein, results in a more enhanced and / or prolonged immune response. An adjuvant can, for example, favor a Th1-predominantly mediated immune response, a Th1-type immune response, or a Th1-mediated immune response. Similarly, an adjuvant can favor a cell-mediated immune response over an antibody-mediated response. Alternatively, an adjuvant can favor an antibody-mediated response. Some adjuvants can enhance the immune response by slowly releasing antigens, while other adjuvants can mediate their effects by one of the following mechanisms: increasing cellular infiltration, inflammation and trafficking to the injection site, particularly for antigen-presenting cells (APCs); promoting an activated state of APCs by upregulating costimulatory signals or major histocompatibility complex (MHC) expression; enhancing antigen presentation; or inducing cytokine release for indirect effects.

[0306] Examples of adjuvants include saponin QS21, CpG oligonucleotides, unmethylated CpG-containing oligonucleotides, MPL, TLR agonists, TLR4 agonists, TLR9 agonists, Resiquimod®, imiquimod, cytokines or nucleic acids encoding them, chemokines or nucleic acids encoding them, IL-12 or nucleic acids encoding them, IL-6 or nucleic acids encoding them, and lipopolysaccharides. Another example of a suitable adjuvant is Montanide ISA 51, which contains a natural metabolizable oil or a purified emulsifier. Other examples of suitable adjuvants include granulocyte / macrophage colony-stimulating factor (GM-CSF) or nucleic acids encoding it, and keyhole limpet hemocyanin (KLH) protein or nucleic acids encoding it. GM-CSF may be a human protein grown in a yeast (S. cerevisiae) vector, for example. GM-CSF promotes the clonal expansion and differentiation of hematopoietic progenitor cells, antigen-presenting cells (APCs), dendritic cells, and T cells.

[0307] Yet another example of a suitable adjuvant is detoxified listeriolysin O (dtLLO) protein. Detoxification can be achieved by introducing point mutations into three selected amino acids that are important for LLO binding to cholesterol and ultimately membrane pore formation. The three targeted amino acids are present in the cholesterol-binding domain of LLO (ECTGLAWEWWR; SEQ ID NO: 74) and can be modified within the sequence by point mutations introduced into the DNA sequence by PCR (EATGLAWEAAR; SEQ ID NO: 96). An example of a dtLLO suitable for use as an adjuvant is encoded by SEQ ID NO: 95. The detoxified, non-hemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and can activate innate and cellular immune responses by acting as a PAMP. dtLLO encoded by a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 95 is also suitable for use as an adjuvant.

[0308] Still other examples of adjuvants include growth factors or nucleic acids encoding same, cell populations, Frond's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG (bacilli Calmette-Guerin), alum, interleukins or nucleic acids encoding same, quill glycoside, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins, or any other type of known adjuvant (e.g., see Fundamental Immunology, 5th ed. (August 2003): William E. Paul, incorporated herein by reference in its entirety and for all purposes). (Editor); Lippincott Williams & Wilkins Publishers; see Chapter 43: Vaccines, GJV Nossal).

[0309] The immunogenic composition may further comprise one or more immunomodulatory molecules. Examples include interferon gamma, cytokines, chemokines, and T-cell stimulators.

[0310] The immunogenic composition may be in the form of a vaccine or pharmaceutical composition. The terms "vaccine" and "pharmaceutical composition" are interchangeable and refer to an immunogenic composition in a pharmaceutically acceptable carrier for in vivo administration to a subject. A vaccine may be, for example, a vaccine contained within and delivered by a cell (e.g., a recombinant Listeria as disclosed herein). A vaccine may prevent a subject from contracting or developing a disease, and / or the vaccine may be therapeutic for a subject with a disease.

[0311] A "pharmaceutically acceptable carrier" refers to a vehicle containing an immunogenic composition that can be introduced into a subject without significant adverse effects and does not have adverse effects on the immunogenic composition. That is, "pharmaceutically acceptable" refers to any formulation that is safe and provides for the delivery of an effective amount of at least one immunogenic composition for use in the methods disclosed herein, suitable for the desired route of administration. Pharmaceutically acceptable carriers, vehicles, or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection can be found in a variety of readily available sources, such as Remington's Pharmaceutical Sciences, 18th ed., 1990, incorporated herein by reference in its entirety for all purposes. Such carriers may be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions can be buffered, e.g., where the pH is maintained at a specific desired value ranging from pH 4.0 to pH 9.0, depending on the stability of the immunogenic composition and the route of administration.

[0312] Suitable pharmaceutically acceptable carriers include, for example, sterile water, saline, e.g., saline, glucose, buffered solutions, e.g., phosphate buffered solutions or bicarbonate buffered solutions, alcohol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, flavor oils, fatty acid mono- and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition or vaccine may also contain auxiliary substances, including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, thickening additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, colorings, flavorings, aromatic substances, etc., which do not deleteriously react with the immunogenic composition.

[0313] For liquid formulations (e.g., in embodiments in which lyophilized recombinant bacteria or Listeria strains are reconstituted by dissolving in a volume of solvent), for example, the pharmaceutically acceptable carrier can be an aqueous or non-aqueous solution, suspension, emulsion, or oil. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions, or suspensions (including saline and buffered media). Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers / diluents include, for example, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, or dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0314] If necessary, sustained-release or triggered-release pharmaceutical compositions or vaccines can be formulated. This formulation can be achieved, for example, by using liposomes or compositions in which the active compound is protected with a gradually degradable coating (e.g., by microencapsulation, multiple coatings, etc.). Such compositions can be formulated for immediate release or sustained release. The compositions can also be freeze-dried, and the resulting lyophilized product can be used (e.g., for preparing injectable products).

[0315] The immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein can comprise one or more additional compounds effective in the prevention or treatment of cancer. For example, the additional compounds can be compounds useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadelimplant, hydroxycarbamide, idarubicin, or ifosfamide. , irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. The additional compounds can also include other biologics, such as Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or antibodies against the EGF receptor, such as Erbitux® (cetuximab) and Vectibix® (panitumumab). The additional compounds can also include, for example, additional immunotherapeutic agents.

[0316] Additional compounds may also include immune checkpoint inhibitor antagonists, such as PD-1 signaling pathway inhibitors, CD-80 / 86 and CTLA-4 signaling pathway inhibitors, T-cell membrane protein 3 (TIM3) signaling pathway inhibitors, adenosine A2a receptor (A2aR) signaling pathway inhibitors, lymphocyte-activation gene 3 (LAG3) signaling pathway inhibitors, killer immunoglobulin receptor (KIR) signaling pathway inhibitors, CD40 signaling pathway inhibitors, or any other antigen-presenting cell / T-cell signaling pathway inhibitors. Examples of immune checkpoint inhibitor antagonists include anti-PD-L1 / PD-L2 antibodies or fragments thereof, anti-PD-1 antibodies or fragments thereof, anti-CTLA-4 antibodies or fragments thereof, or anti-B7-H4 antibodies or fragments thereof. Additional compounds may also include T-cell stimulators, such as antibodies or functional fragments thereof that bind to T-cell receptor costimulatory molecules, antigen-presenting cell receptors that bind to costimulatory molecules, or members of the TNF receptor superfamily. T cell receptor costimulatory molecules may include, for example, CD28 or ICOS. Antigen-presenting cell receptors that bind to costimulatory molecules may include, for example, CD80 receptors, CD86 receptors, or CD46 receptors. TNF receptor superfamily members may include, for example, glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25. See, for example, WO2016100929, WO2016011362, and WO2016011357, each of which is incorporated herein by reference in its entirety for all purposes. VI. Treatment method

[0317] The lyophilized bacteria or Listeria strains (if necessary, the lyophilized recombinant bacteria or Listeria strains are reconstituted by dissolving them in a certain amount of solvent), immunogenic compositions, pharmaceutical compositions, and vaccines disclosed herein can be used in various methods. For example, they can be used in methods for inducing or enhancing an anti-disease-associated antigen (e.g., cancer-associated antigen or tumor-associated antigen) immune response in a subject, inducing or enhancing an anti-disease (e.g., anti-tumor or anti-cancer) immune response in a subject, treating a disease (e.g., tumor or cancer) in a subject, preventing a disease (e.g., tumor or cancer) in a subject, or protecting a subject from a disease (e.g., tumor or cancer). They can also be used in methods for increasing the ratio of effector T cells to regulatory T cells (Tregs) in a subject's spleen and tumor, where the effector T cells are targeted to a disease-associated antigen. They may also be used in methods of increasing disease-associated antigen T cells in a subject, prolonging survival of a subject with a disease, delaying the onset of disease in a subject, or alleviating symptoms of a disease in a subject.

[0318] A method for inducing or enhancing an anti-disease-associated antigen immune response in a subject can include, for example, administering to the subject a lyophilized or reconstituted recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine disclosed herein. This can induce or enhance an anti-disease-associated antigen immune response in the subject. For example, in the case of a lyophilized or reconstituted recombinant Listeria strain, the Listeria strain can express a fusion polypeptide, thereby eliciting an immune response in the subject. The immune response can include, for example, a T cell response, such as a CD4+FoxP3- T cell response, a CD8+ T cell response, or a CD4+FoxP3- and CD8+ T cell response. Such a method can also increase the ratio of effector T cells to regulatory T cells (Tregs) in the subject's spleen and tumor microenvironment, resulting in a more pronounced anti-tumor response in the subject.

[0319] A method for inducing or enhancing an anti-disease (e.g., anti-cancer or anti-tumor) immune response in a subject can include, for example, administering to the subject a lyophilized or reconstituted recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine disclosed herein. This can induce or enhance an anti-disease immune response in the subject. For example, in the case of a recombinant Listeria strain, the Listeria strain can express a fusion polypeptide, thereby eliciting a disease-associated response in the subject.

[0320] A method of treating a disease (e.g., cancer or tumor) in a subject can include, for example, administering to the subject a lyophilized or reconstituted recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine disclosed herein, such that the subject can mount an immune response against a disease that expresses a disease-associated antigen, thereby treating the disease in the subject.

[0321] A method of preventing a disease (e.g., cancer or tumor) in a subject or protecting a subject from developing a disease can include, for example, administering to a subject a lyophilized or reconstituted recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine disclosed herein, such that the subject can mount an immune response to disease-associated antigens, thereby preventing the disease or protecting the subject from developing the disease.

[0322] In some of the above methods, two or more lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered. Multiple recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered sequentially in any order or combination, or simultaneously in any combination. For example, if four different Listeria strains are to be administered, they can be administered sequentially, simultaneously, or in any combination (e.g., administering the first and second strains simultaneously, followed by administering the third and fourth strains simultaneously). Optionally, in the case of sequential administration, the compositions can be administered during the same immune response. In some embodiments, the compositions are administered within 0 to 10 or 3 to 7 days of each other. Multiple recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can each contain a different set of antigenic peptides. Alternatively, two or more can contain the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[0323] In some embodiments, the disease is cancer or a tumor. Cancer is a physiological condition in mammals typically characterized by unregulated cell growth and proliferation. Cancer can also be a hematopoietic malignancy or a solid tumor (i.e., a mass of cells resulting from excessive cell growth and proliferation, including precancerous lesions). Metastatic cancer refers to a cancer that has spread from the original site of initiation to another location in the body. Tumors formed by metastatic cancer cells are called metastatic tumors or metastases, a term also used to refer to the process by which cancer cells spread to other parts of the body. Generally, metastatic cancers have the same name and type of cancer cells as the original, or primary, cancer. Examples of solid tumors include melanoma, carcinoma, blastoma, and sarcoma. Hematological malignancies include, for example, leukemia or lymphoid malignancies, such as lymphoma. Exemplary cancer categories include brain cancer, breast cancer, gastrointestinal cancer, genitourinary cancer, gynecological cancer, head and neck cancer, hemocytic cancer, skin cancer, and chest cancer. Brain malignancies include, for example, glioblastoma, high-grade pontine glioma, low-grade glioma, medulloblastoma, neuroblastoma, and pilocytic astrocytoma. Gastrointestinal cancers include, for example, colorectal cancer, gallbladder cancer, hepatocellular carcinoma, pancreatic cancer, PNET, gastric cancer, and esophageal cancer. Genitourinary cancers include, for example, adrenocortical carcinoma, bladder cancer, chromophobe kidney cancer, renal (clear cell) carcinoma, renal (papillary) carcinoma, rhabdoid carcinoma, and prostate cancer. Gynecological cancers include, for example, uterine carcinosarcoma, endometrial cancer, serous ovarian cancer, and cervical cancer. Head and neck cancers include, for example, thyroid cancer, nasopharyngeal cancer, head and neck cancer, and adenoid cystic carcinoma. Hemolytic cancers include, for example, multiple myeloma, myelodysplasia, mantle cell lymphoma, acute lymphocytic leukemia (ALL), non-lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML). Skin cancers include, for example, cutaneous melanoma and squamous cell carcinoma. Thoracic cancers include, for example, squamous cell lung cancer, small cell lung cancer, and lung adenocarcinoma.

[0324] More specific examples of such cancers include squamous cell carcinoma or carcinoma (e.g., oral squamous cell carcinoma), myeloma, oral cancer, juvenile nasopharyngeal angiofibroma, neuroendocrine tumors, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, glial tumors, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer or carcinoma, salivary gland cancer, kidney or renal cancer (e.g., renal cell carcinoma), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, fibrosarcoma, gallbladder cancer, osteosarcoma, mesothelioma, and head and neck cancer. The cancer may also be brain cancer or another type of CNS or intracranial tumor. For example, the subject may have an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma), an oligodendroglial tumor (e.g., oligodendroglioma, anaplastic oligodendroglioma), an ependymal cell tumor (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), a mixed glioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma), a neuroepithelial tumor of unknown etiology (e.g., polar spongioblastoma, astroblastoma, gliomatosis cerebri), a tumor of the choroid plexus (e.g., , choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixed neuronal-glial tumors (e.g., gangliocytoma, cerebellar dysplastic gangliocytoma, ganglioglioma, anaplastic ganglioglioma, desmoplastic infantile ganglioneuroma, central neurocytoma, dysembryoplastic neuroepithelial tumor, olfactory neuroblastoma), pineal parenchymal tumors (e.g., pineocytoma, pineoblastoma, mixed pineocytoma / pineoblastoma), or tumors with mixed neuronal or neuroblastic elements (e.g., medulloepithelioma, glioblastoma, neuroblastoma, retinoblastoma, ependymoblastoma).

[0325] The term "treat" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or alleviate the symptoms of the target disease. Treating can include one or more of directly influencing or curing the disease, suppressing, inhibiting, preventing, reducing the severity of the disease, delaying the onset of the disease, slowing the progression of the disease, stabilizing the progression of the disease, inducing remission of the disease, preventing or delaying metastasis of the disease, and alleviating / ameliorating symptoms associated with the disease, or a combination thereof. For example, treating can include extending expected survival. The effect (e.g., suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, inducing remission of, preventing or delaying, alleviating / ameliorating symptoms of, etc.) can be compared to a control subject not receiving treatment or receiving a placebo. The term "treat" or "treating" can also refer to increasing the percent chance of survival or extending the expected survival time for a subject with a disease (e.g., compared to a control subject not receiving treatment or receiving a placebo). In one example, "treating" refers to delaying progression (e.g., compared to a control subject not receiving treatment or receiving a placebo), promoting remission, inducing remission, increasing remission, accelerating recovery, increasing the effectiveness of alternative therapies, reducing resistance to alternative therapies, or a combination thereof. The term "preventing" or "preventing" can refer, for example, to delaying the onset of symptoms, preventing recurrence of disease, reducing the number or frequency of recurrent episodes, extending the latency period between symptomatic episodes, or a combination thereof.The terms "suppressing" or "inhibiting" can refer, for example, to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, shortening the latency period of symptoms, ameliorating symptoms, alleviating secondary symptoms, reducing secondary infections, increasing patient survival, or a combination thereof.

[0326] The term "subject" refers to a mammal (e.g., a human) in need of treatment for or susceptible to developing a disease. The term subject also refers to a mammal (e.g., a human) receiving either prophylactic or therapeutic treatment. Subjects can include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude individuals who are healthy in all respects and who do not have or show signs of disease.

[0327] An individual is at high risk of developing a disease if the subject has at least one risk factor (e.g., genetic, biochemical, family history, and situational exposure) that is known to place an individual with that risk factor at a statistically significantly higher risk of developing the disease than an individual without that risk factor.

[0328] A "symptom" or "sign" refers to objective evidence of disease observed by a physician or subjective evidence of disease perceived by a subject, e.g., a change in gait. A symptom or sign can be any manifestation of disease. A symptom can be primary or secondary. The term "primary" refers to a symptom that is a direct result of a specific disease or disorder (e.g., a tumor or cancer), while the term "secondary" refers to a symptom that originates from or results from a primary cause. The lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein can treat primary or secondary symptoms or secondary complications.

[0329] The lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine is administered in an effective regimen, where an effective regimen refers to a dosage, route of administration, and frequency of administration that delays the onset of, reduces the severity of, inhibits further progression of, and / or ameliorates at least one sign or symptom of the disease. Alternatively, the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine is administered in an effective regimen, where an effective regimen refers to a dosage, route of administration, and frequency of administration that induces an immune response to disease-associated antigens in the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine, or to the bacteria or Listeria strain itself. If the subject already has the disease, the regimen can be referred to as a therapeutically effective regimen. If the subject is at increased risk of developing the disease compared to the general population but has not yet experienced symptoms, the regimen can be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in individual patients compared with historical controls or compared with past experience in the same patient.In other cases, therapeutic or prophylactic efficacy can be demonstrated in a group of treated patients in preclinical or clinical trials compared with a control group of untreated patients.For example, if individual patients treated by the methods described herein achieve a more favorable outcome than the average outcome in a control group of untreated comparable patients, or if a more favorable outcome is demonstrated in treated patients compared with control patients in controlled clinical trials (e.g., Phase II, Phase II / III, or Phase III trials) at a p<0.05, or 0.01, or even 0.001 level, the regimen can be considered therapeutically or prophylactically effective.

[0330] An exemplary dosage for a recombinant Listeria strain is, for example, 1 x 10 6 ~1×10 7 CFU, 1 × 10 7 ~1×10 8 CFU, 1 × 108 ~3.31×10 10 CFU, 1 × 10 9 ~3.31×10 10 CFU, 5–500 × 10 8 CFU, 7–500 × 10 8 CFU, 10–500 × 10 8 CFU, 20–500 × 10 8 CFU, 30–500 × 10 8 CFU, 50–500 × 10 8 CFU, 70–500 × 10 8 CFU, 100–500 × 10 8 CFU, 150-500 x 10 8 CFU, 5–300 × 10 8 CFU, 5–200 × 10 8 CFU, 5–15 × 10 8 CFU, 5–100 × 10 8 CFU, 5–70 × 10 8 CFU, 5–50 × 10 8 CFU, 5–30 × 10 8 CFU, 5–20 × 10 8 CFU, 1–30 × 10 9 CFU, 1–20 × 10 9 CFU, 2–30 × 10 9 CFU, 1–10 × 10 9 CFU, 2–10 × 10 9 CFU, 3–10 × 10 9 CFU, 2–7 × 10 9 CFU, 2–5 × 10 9 CFU, and 3–5 × 10 9 Other exemplary dosages for recombinant Listeria strains are, for example, 1 x 10 CFU. 7 Biology, 1.5×10 7 Biology, 2×10 8 Biology, 3×10 7 Biology, 4×10 7 Biology, 5×10 7 Biology, 6×10 7 Biology, 7×10 7 Biology, 8×10 7 Biology, 10×10 7 Biology, 1.5×10 8 Biology, 2×10 8Biology, 2.5×10 8 Biology, 3×10 8 Biology, 3.3×10 8 Biology, 4×10 8 Biology, 5×10 8 Biology, 1×10 9 Biology, 1.5×10 9 Biology, 2×10 9 Biology, 3×10 9 Biology, 4×10 9 Biology, 5×10 9 Biology, 6×10 9 Biology, 7×10 9 Biology, 8×10 9 Biology, 10×10 9 Biology, 1.5×10 10 Biology, 2×10 10 Biology, 2.5×10 10 Biology, 3×10 10 Biology, 3.3×10 10 Biology, 4×10 10 Biology, and 5x10 10 Dosage may depend on the patient's condition, response to previous treatment, if any, whether that treatment was prophylactic or therapeutic, and other factors.

[0331] Administration can be by any suitable means. For example, administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraventricular, intraperitoneal, topical, intranasal, intramuscular, intraocular, intrarectal, conjunctival, transdermal, intradermal, vaginal, rectal, intratumoral, paracanthal, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal aspirate (spray), sublingual, aerosol, suppository, or a combination thereof. For intranasal administration or application by inhalation, a solution or suspension of the recombinant fusion polypeptide, nucleic acid encoding the recombinant fusion polypeptide, recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine mixed in the presence of a suitable carrier and aerosolized or atomized is suitable. Such aerosols can contain any of the lyophilized or reconstituted recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine described herein. Administration can be in the form of suppository (for example, rectal suppository or urethral suppository), pellet form for subcutaneous implantation (for example, to provide controlled release over a period of time), or capsule form.Administration can also be by injection into disease site.Dosage regimen can be easily determined based on factors such as the exact nature and type of disease to be treated, the severity of disease, the age and general health condition of the subject, the weight of the subject, the response of each individual subject, etc.

[0332] The frequency of administration may depend on, among other factors, the half-life of the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine in the subject, the condition of the subject, and the route of administration. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals, for example, depending on changes in the subject's condition or the progression of the tumor or cancer under treatment. The treatment course may depend on the subject's condition and other factors. For example, the treatment course may last for several weeks, months, or years (e.g., up to two years). Repeat administration (doses) may begin immediately after the initial treatment course or several days, weeks, or months apart, for example, to achieve regression or suppression of the disease. Assessment may be determined by any known technique, including diagnostic methods, such as imaging techniques, serum biomarkers, biopsies, or analysis of the presence, absence, or improvement of disease-related symptoms. As a specific example, the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine may be administered once every three weeks for up to two years. In one example, the freeze-dried or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines disclosed herein are administered in increasing doses to increase the ratio of effector T cells to regulatory T cells, and generate a stronger anti-disease immune response.For example, cytokines, including IFN-γ, TNF-α, and other cytokines known to enhance cellular immune response, can be provided to the subject to further enhance the anti-disease response.See, for example, US6,991,785, the entire contents of which are incorporated herein by reference for all purposes.

[0333] Some methods may further include "boosting" the subject with additional lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines, or administering multiple doses of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines. "Boosting" refers to administering additional doses to the subject. For example, in some methods, two boosters (or a total of three inoculations) are administered, three boosters are administered, four boosters are administered, five boosters are administered, or six or more boosters are administered. The number of doses administered may depend, for example, on the response of the disease to the treatment.

[0334] Optionally, the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the booster inoculation is the same as the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the initial "priming" inoculation. Alternatively, the booster is different from the priming recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. Optionally, the same dosage is used in the priming and booster inoculations. Alternatively, a higher dosage is used for the booster, or a lower dosage is used for the booster. The period between the priming and booster inoculations can be determined empirically. For example, the period between the priming and booster inoculations can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks.

[0335] Heterologous prime-boost strategies have been effective in enhancing immune responses and protection against numerous pathogens. See, e.g., Schneider et al. (1999) Immunol. Rev. 170:29-38; Robinson (2002) Nat. Rev. Immunol. 2:239-250; Gonzalo et al. (2002) Vaccine 20:1226-1231; and Tanghe (2001) Infect. Immun. 69:3041-3047, each of which is incorporated herein by reference in its entirety for all purposes. Providing different forms of antigen in the prime and boost injections can maximize the immune response to the antigen. Priming with a DNA vaccine followed by boosting with a protein in an adjuvant or viral vector delivery of DNA encoding the antigen can increase antigen-specific antibodies and CD4 + T cell response or CD8 + This is an effective method for improving T cell responses. See, for example, Shiver et al. (2002) Nature 415: 331-335; Gilbert et al. (2002) Vaccine 20:1039-1045; Billaut-Mulot et al. (2000) Vaccine 19:95-102; and Sin et al. (1999) DNA Cell Biol.18:771-779, each of which is incorporated herein by reference in its entirety for all purposes. For example, when a subject is vaccinated by DNA priming followed by boosting with an adenoviral vector expressing an antigen, the T cell response can be enhanced by adding CRL1005 poloxamer (12 kDa, 5% POE) to the DNA encoding the antigen. For example, see Shiver et al. (2002) Nature 415:331-335, the entirety of which is incorporated herein by reference for all purposes. As another example, a vector construct encoding an immunogenic portion of an antigen and a protein containing the immunogenic portion of an antigen can be administered. See, e.g., US2002 / 0165172, which is incorporated herein by reference in its entirety for all purposes. Similarly, the immune response to nucleic acid vaccination can be enhanced by co-administration of a polynucleotide and a polypeptide of interest (e.g., during the same immune response, in some embodiments, within 0-10 or 3-7 days of each other). See, e.g., US6,500,432, which is incorporated herein by reference in its entirety for all purposes.

[0336] The therapeutic methods disclosed herein can also comprise administering one or more additional compounds effective in the prevention or treatment of disease (e.g., tumor or cancer).For example, the additional compounds can be compounds useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadel implant, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, These may include leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Alternatively, the additional compound can include other biologics, such as Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or antibodies against the EGF receptor, such as Erbitux® (cetuximab) and Vectibix® (panitumumab). Alternatively, the additional compound can include other immunotherapeutic agents. Alternatively, the additional compound can be an indoleamine 2,3-dioxygenase (IDO) pathway inhibitor, such as 1-methyltryptophan (1MT), 1-methyltryptophan (1MT), necrostatin-1, pyridoxal isonicotinoyl hydrazone, ebselen, 5-methylindole-3-carboxaldehyde, CAY10581, an anti-IDO antibody, or a small molecule IDO inhibitor.IDO inhibition can enhance the effectiveness of chemotherapeutic agents. The therapeutic methods disclosed herein can also be combined with radiation, stem cell treatment, surgery, or any other treatment.

[0337] Such additional compounds or treatments may precede administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines disclosed herein, may follow administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines disclosed herein, or may be concurrent with administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines disclosed herein.

[0338] Targeted immunomodulatory therapy primarily focuses on activating costimulatory receptors, for example, by using agonistic antibodies targeting members of the tumor necrosis factor receptor superfamily, including 4-1BB, OX40, and GITR (glucocorticoid-inducible TNF receptor-related). Modulation of GITR has demonstrated potential in both anti-tumor and vaccine settings. Another target for agonistic antibodies is costimulatory signaling molecules for T cell activation. Targeting costimulatory signaling molecules can result in enhanced T cell activation and foster a more potent immune response. Costimulation can also help prevent the inhibitory effects of checkpoint blockade and increase antigen-specific T cell proliferation.

[0339] Listeria-based immunotherapy works by inducing the de novo generation of tumor antigen-specific T cells that infiltrate and destroy tumors, as well as by reducing the number and activity of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Antibodies (or functional fragments thereof) against T cell co-inhibitory or co-stimulatory receptors (e.g., checkpoint inhibitors CTLA-4, PD-1, TIM-3, LAG3 and co-stimulators CD137, OX40, GITR, and CD40) can have synergistic effects with Listeria-based immunotherapy.

[0340] Thus, some methods may further comprise administering a composition comprising an immune checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80 / 86 and CTLA-4 signaling pathway inhibitor, a T-cell membrane protein 3 (TIM3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte-activation gene 3 (LAG3) signaling pathway inhibitor, a killer immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen-presenting cell / T-cell signaling pathway inhibitor. Examples of immune checkpoint inhibitor antagonists include an anti-PD-L1 / PD-L2 antibody or fragment thereof, an anti-PD-1 antibody or fragment thereof, an anti-CTLA-4 antibody or fragment thereof, or an anti-B7-H4 antibody or fragment thereof. For example, the anti-PD-1 antibody can be administered to a subject at 5-10 mg / kg once every two weeks, 5-10 mg / kg once every three weeks, 1-2 mg / kg once every three weeks, 1-10 mg / kg once every week, 1-10 mg / kg once every two weeks, 1-10 mg / kg once every three weeks, or 1-10 mg / kg once every four weeks.

[0341] Similarly, some methods may further include administering a T cell stimulatory substance, such as an antibody or functional fragment thereof that binds to a T cell receptor costimulatory molecule, an antigen-presenting cell receptor that binds to a costimulatory molecule, or a member of the TNF receptor superfamily. T cell receptor costimulatory molecules may include, for example, CD28 or ICOS. Antigen-presenting cell receptors that bind to costimulatory molecules may include, for example, CD80 receptor, CD86 receptor, or CD46 receptor. TNF receptor superfamily members may include, for example, glucocorticoid-inducible TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25.

[0342] For example, some methods can further include administering an effective amount of a composition comprising an antibody or functional fragment thereof that binds to a T cell receptor costimulatory molecule, or an antibody or functional fragment thereof that binds to an antigen-presenting cell receptor that binds to a costimulatory molecule. The antibody can be, for example, an anti-TNF receptor antibody or antigen-binding fragment thereof (e.g., TNF receptor superfamily member glucocorticoid-inducible TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25), an anti-OX40 antibody or antigen-binding fragment thereof, or an anti-GITR antibody or antigen-binding fragment thereof. Alternatively, other agonistic molecules (e.g., GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an antigen-presenting cell (APC) / T cell agonist, CD134 or a ligand or fragment thereof, CD137 or a ligand or fragment thereof, or inducible T cell costimulatory molecule (ICOS) or a ligand or fragment thereof, or an agonistic small molecule) can be administered.

[0343] In certain examples, some methods can further include administering an anti-CTLA-4 antibody or functional fragment thereof and / or an anti-CD137 antibody or functional fragment thereof. For example, the anti-CTLA-4 antibody or functional fragment thereof or the anti-CD137 antibody or functional fragment thereof can be administered about 72 hours after the first dose of the recombinant fusion polypeptide, nucleic acid encoding the recombinant fusion polypeptide, recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine, or about 48 hours after the first dose of the recombinant fusion polypeptide, nucleic acid encoding the recombinant fusion polypeptide, recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. The anti-CTLA-4 antibody or functional fragment thereof or the anti-CD137 antibody or functional fragment thereof can be administered, for example, at a dose of about 0.05 mg / kg and about 5 mg / kg. The recombinant Listeria strain or immunogenic composition comprising the recombinant Listeria strain can be administered, for example, at a dose of about 1 x 10 9 CFU. Some such methods may further comprise administering an effective amount of an anti-PD-1 antibody or functional fragment thereof.

[0344] Methods for assessing the efficacy of cancer immunotherapy are well known, and are described, for example, in Dzojic et al. (2006) Prostate 66(8):831-838; Naruishi et al. (2006) Cancer Gene Ther. 13(7):658-663; Sehgal et al. (2006) Cancer Cell Int. 6:21), and Heinrich et al. (2007) Cancer Immunol Immunother 56(5):725-730, each of which references is incorporated herein by reference in its entirety for all purposes. By way of example, for prostate cancer, the methods and compositions disclosed herein can be tested using a prostate cancer model, such as the TRAMP-C2 mouse model, the 178-2 BMA cell model, the PAIII adenocarcinoma cell model, the PC-3M model, or any other prostate cancer model.

[0345] Alternatively, or in addition, immunotherapies can be tested in human subjects, and efficacy can be monitored using known methods. Such methods can include, for example, directly measuring CD4+ and CD8+ T cell responses, or measuring disease progression (e.g., by determining the number or size of tumor metastases, or by monitoring disease symptoms, such as coughing, chest pain, weight loss, etc.). Methods for assessing the efficacy of cancer immunotherapies in human subjects are well known and are described, for example, in Uenaka et al. (2007) Cancer Immun. 7:9 and Thomas-Kaskel et al. (2006) Int J Cancer 119(10):2428-2434, each of which is incorporated herein by reference in its entirety for all purposes. VII. Kit

[0346] Kits containing reagents for use in carrying out the methods disclosed herein, or kits containing the compositions, tools or apparatus disclosed herein are also provided.

[0347] For example, such kits may include a lyophilized recombinant bacterium or Listeria strain disclosed herein, an immunogenic composition disclosed herein, a pharmaceutical composition disclosed herein, or a vaccine disclosed herein. Such kits may also include a solvent or diluent for reconstituting the lyophilized recombinant bacterium or Listeria strain. Additionally, such kits may further include instructional materials describing the use of the lyophilized recombinant bacterium or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine to perform the methods disclosed herein. Such kits may further include an applicator, if desired. A model kit is described below, but other useful kit components will be apparent in light of this disclosure.

[0348] All patent applications, websites, other publications, accession numbers, etc., cited above or below, are incorporated by reference in their entirety for all purposes, and this incorporation is to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Even if different versions of a sequence were linked to an accession number at different times, the version linked to that accession number as of the effective filing date of this application is intended. This effective filing date means the earlier of the actual filing date or the filing date of a priority application, if applicable, with reference to the accession number. Similarly, even if different versions of publications, websites, etc. were published at different times, the version published closest to the effective filing date of this application is intended, unless otherwise indicated. Unless specifically indicated otherwise, any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other. While the invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims. Enumeration of Embodiments

[0349] The subject matter disclosed herein includes, but is not limited to, the following embodiments.

[0350] 1. A method for producing a lyophilized composition comprising a Listeria strain, comprising the steps of: (a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose; (b) cooling the composition provided in step (a) at a holding temperature of between about -32°C and about -80°C in a freezing step; (c) exposing the composition produced by step (b) to a vacuum at a holding temperature of between about -10°C and about -30°C in a primary drying step; and (d) exposing the composition produced by step (c) to a vacuum at a holding temperature of between about -5°C and about 25°C in a secondary drying step, thereby producing a lyophilized composition.

[0351] 2. The method of embodiment 1, wherein prior to step (a), a stress response is induced in the Listeria strain by exposing the Listeria strain to a reduced temperature.

[0352] 3. The method of embodiment 1, wherein prior to step (a), a stress response is not induced in the Listeria strain by exposing the Listeria strain to a reduced temperature.

[0353] 4. The method of any preceding embodiment, wherein the Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a).

[0354] 5. The concentration of the frozen Listeria strain to be thawed is about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 5. The method of embodiment 4, wherein the colony forming units (CFUs) are between.

[0355] 6. The method of embodiment 4 or 5, wherein the frozen Listeria strain is thawed at about 2°C to about 37°C.

[0356] 7. The method of embodiment 6, wherein the frozen Listeria strain is thawed at about 20°C to about 37°C.

[0357] 8. The method of embodiment 7, wherein the frozen Listeria strains are thawed at about 32°C and about 37°C.

[0358] 9. The method of embodiment 8, wherein the frozen Listeria strain is thawed at about 37°C.

[0359] 10. The method of any one of embodiments 4 to 9, wherein the frozen Listeria strain is thawed for 8 hours or less.

[0360] 11. The method of any one of embodiments 4 to 10, wherein the frozen Listeria strain is kept at about 2°C to about 8°C for no more than 24 hours after thawing.

[0361] 12. The method of any one of embodiments 1 to 3, wherein the Listeria strain used in the composition in step (a) is freshly cultured prior to step (a).

[0362] 13. The method of any preceding embodiment, wherein the buffer is a phosphate buffer.

[0363] 14. The method of any preceding embodiment, wherein the formulation comprises about 1% to about 5% w / v sucrose.

[0364] 15. The method of embodiment 14, wherein the formulation comprises about 2% to about 3% w / v sucrose.

[0365] 16. The method of embodiment 15, wherein the formulation comprises about 2.5% w / v sucrose.

[0366] 17. The formulation contains approximately 1 x 10 per milliliter 9 ~Approx. 1×10 1010. The method of any preceding embodiment, comprising colony forming units (CFU) of Listeria.

[0367] 18. The method of any preceding embodiment, wherein the formulation does not include one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

[0368] 19. The method of embodiment 18, wherein the formulation does not contain trehalose, MSG, or rHSA.

[0369] 20. The method of any preceding embodiment, wherein the holding temperature in the freezing step (b) is between about -40°C and about -50°C.

[0370] 21. The method of embodiment 20, wherein the holding temperature in the freezing step (b) is about -45°C.

[0371] 22. The method of any preceding embodiment, wherein the freezing step (b) comprises decreasing the temperature to the holding temperature at a rate of about 1° C. per minute.

[0372] 23. The method of any preceding embodiment, wherein the cooling in the freezing step (b) is for about 2 hours to about 4 hours.

[0373] 24. The method of any preceding embodiment, wherein the cooling in the freezing step (b) comprises holding the composition at the holding temperature for about 2 hours.

[0374] 25. The method of any preceding embodiment, wherein the holding temperature in the primary drying step (c) is between about -12°C and about -22°C.

[0375] 26. The method of embodiment 25, wherein the holding temperature in the primary drying step (c) is between about -17°C and about -19°C.

[0376] 27. The method of embodiment 26, wherein the holding temperature in the primary drying step (c) is about -18°C.

[0377] 28. The method of any preceding embodiment, wherein the primary drying step (c) comprises increasing the temperature to the holding temperature at a rate of about 1° C. per minute.

[0378] 29. The method of any preceding embodiment, wherein the primary drying step (c) is for about 25 hours to about 35 hours.

[0379] 30. The method of any preceding embodiment, wherein the primary drying step (c) is terminated about 12 to about 16 hours after the composition has reached the holding temperature.

[0380] 31. The method of any preceding embodiment, wherein the primary drying step (c) is at a vacuum pressure of about 0.09 mbar.

[0381] 32. The method of any preceding embodiment, wherein the holding temperature in the secondary drying step (d) is between about -5°C and about 20°C.

[0382] 33. The method of embodiment 32, wherein the holding temperature in the secondary drying step (d) is between about -5°C and about 5°C.

[0383] 34. The method of embodiment 33, wherein the holding temperature in the secondary drying step (d) is about 0°C.

[0384] 35. The method of any preceding embodiment, wherein the secondary drying step (d) comprises increasing the temperature to the holding temperature at a rate of about 0.2°C per minute.

[0385] 36. The method of any preceding embodiment, wherein the secondary drying step (d) is for about 1 hour to about 10 hours.

[0386] 37. The method of any preceding embodiment, wherein the secondary drying step (d) comprises holding the composition at the holding temperature for about 2 hours to about 6 hours.

[0387] 38. The method of embodiment 37, wherein the secondary drying step (d) comprises holding the composition at the holding temperature for about 5 hours to about 6 hours.

[0388] 39. The method of any preceding embodiment, wherein the secondary drying step (d) is at a vacuum pressure of about 0.09 mbar.

[0389] 40. The method of any preceding embodiment, wherein the residual moisture in the lyophilized composition is between about 1% and about 5%.

[0390] 41. The method of embodiment 40, wherein the residual moisture in the lyophilized composition is between about 2% and about 4%.

[0391] 42. The method of any preceding embodiment, wherein the residual moisture in the lyophilized composition is at least about 2.5%.

[0392] 43. The method of embodiment 42, wherein the residual moisture in the lyophilized composition is at least about 3%.

[0393] 44. The method of any preceding embodiment, wherein the lyophilized composition exhibits a viability of at least about 60% after storage between about -20°C and about 4°C for about 12 months.

[0394] 45. The method of embodiment 44, wherein the freeze-dried composition exhibits a viability of at least about 75% after storage between about -20°C and about 4°C for about 12 months.

[0395] 46. ​​The method of embodiment 45, wherein the freeze-dried composition exhibits a viability of at least about 80% after storage at between about -20°C and about 4°C for about 12 months.

[0396] 47. The method of any preceding embodiment, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

[0397] 48. The Listeria strain is a recombinant Listeria monocytogenes strain, the buffer is a phosphate buffer, the formulation contains about 2% to about 3% w / v sucrose, the formulation does not contain trehalose, MSG, or rHSA, and the formulation contains about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 The method of any preceding embodiment, wherein the freeze-dried composition contains colony forming units (CFU) of Listeria, the holding temperature in the freezing step (a) is between about -40°C and about -50°C, the holding temperature in the primary drying step (c) is between -17°C and -19°C, and the holding temperature in the secondary drying step (d) is between -1°C and 1°C, and the residual moisture in the freeze-dried composition is between about 2.5% and about 4%.

[0398] 49. The Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a), and the thawed frozen Listeria strain has a concentration of about 1 x 10 per milliliter. 9 ~Approx. 1×10 10 49. The method of claim 48, wherein the frozen Listeria strain is between colony forming units (CFU), the frozen Listeria strain is thawed at about 37°C, the frozen Listeria strain is thawed for 8 hours or less, and the frozen Listeria strain is kept at about 2°C to about 8°C for 24 hours or less after thawing.

[0399] 50. The method of any preceding embodiment, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, and the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

[0400] 51. The method of embodiment 50, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

[0401] 52. The method of embodiment 50, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain containing deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and contains a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO.

[0402] 53. A formulation for lyophilization of a Listeria strain, comprising: (1) a Listeria strain; (2) a phosphate buffer; and (3) sucrose.

[0403] 54. The formulation of embodiment 53, wherein the Listeria strain is a strain in which a stress response has been induced by exposing the Listeria strain to a reduced temperature.

[0404] 55. The formulation of embodiment 53 or 54, wherein the Listeria strain is from a frozen Listeria stock.

[0405] 56. The formulation of embodiment 53 or 54, wherein the Listeria strain is from a freshly cultured Listeria stock.

[0406] 57. A formulation described in any one of embodiments 53 to 56, comprising about 1% to about 5% w / v sucrose.

[0407] 58. The formulation of embodiment 57, comprising about 2% to about 3% w / v sucrose.

[0408] 59. The formulation of embodiment 58, comprising about 2.5% w / v sucrose.

[0409] 60. The formulation of any one of embodiments 53-59, which does not contain one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

[0410] 61. The formulation of embodiment 60, which does not contain trehalose, MSG, or rHSA.

[0411] 62. The formulation of any one of embodiments 53 to 61, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

[0412] 63. A formulation described in any one of embodiments 53-62, wherein the Listeria strain is a recombinant Listeria monocytogenes strain, the formulation comprises about 2% to about 3% w / v sucrose, and the formulation does not comprise trehalose, MSG, or rHSA.

[0413] 64. The formulation of any one of embodiments 53-63, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, and the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

[0414] 65. The formulation of embodiment 64, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

[0415] 66. The formulation of embodiment 64, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO.

[0416] 67. A lyophilized composition produced by a method according to any one of embodiments 1 to 52.

[0417] 68. A lyophilized composition comprising a Listeria strain, a phosphate buffer, and sucrose.

[0418] 69. The lyophilized composition of embodiment 68, which does not contain one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

[0419] 70. The lyophilized composition of embodiment 69, which does not contain trehalose, MSG, or rHSA.

[0420] 71. A freeze-dried composition described in any one of embodiments 67 to 70, wherein the residual moisture in the freeze-dried composition is between about 1% and about 5%.

[0421] 72. The freeze-dried composition of embodiment 71, wherein the residual moisture in the freeze-dried composition is between about 2% and about 4%.

[0422] 73. A freeze-dried composition described in any one of embodiments 67 to 72, wherein the residual moisture in the freeze-dried composition is at least about 2.5%.

[0423] 74. The freeze-dried composition of embodiment 73, wherein the residual moisture in the freeze-dried composition is at least about 3%.

[0424] 75. A freeze-dried composition comprising a Listeria strain, wherein the residual moisture in the freeze-dried composition is at least about 2.5%.

[0425] 76. A freeze-dried composition described in any one of embodiments 67 to 75, exhibiting a viability of at least about 60% after storage at between about -20°C and about 4°C for about 12 months.

[0426] 77. The freeze-dried composition of embodiment 76, which exhibits a viability of at least about 75% after storage at between about -20°C and about 4°C for about 12 months.

[0427] 78. The freeze-dried composition of embodiment 77, which exhibits a viability of at least about 80% after being stored at between about -20°C and about 4°C for about 12 months.

[0428] 79. The freeze-dried composition of any one of embodiments 67 to 78, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

[0429] 80. The freeze-dried composition of any one of embodiments 67 to 79, wherein the Listeria strain is a recombinant Listeria monocytogenes strain, the freeze-dried composition does not contain trehalose, MSG, or rHSA, and the residual moisture in the freeze-dried composition is between 2.5% and 4%.

[0430] 81. The lyophilized composition of any one of embodiments 67 to 80, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, and the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

[0431] 82. The freeze-dried composition of embodiment 81, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

[0432] 83. The freeze-dried composition of embodiment 81, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO.

[0433] 84. A method for preparing frozen Listeria strains for lyophilization, the method comprising thawing the frozen Listeria strains at a temperature between about 20°C and about 37°C.

[0434] 85. The method of embodiment 84, wherein the temperature is between about 32°C and about 37°C.

[0435] 86. The method of embodiment 85, wherein the temperature is about 37°C.

[0436] 87. The method of any one of embodiments 84 to 86, wherein the frozen Listeria strain is thawed for 8 hours or less.

[0437] 88. The method of any one of embodiments 84 to 87, wherein the frozen Listeria strain is kept at about 2°C to about 8°C for no more than 24 hours after thawing.

[0438] 89. The method of any one of embodiments 84 to 88, wherein the frozen Listeria strain is thawed in a formulation comprising a buffer and sucrose.

[0439] 90. The method of embodiment 89, wherein the formulation comprises about 1% to about 5% w / v sucrose.

[0440] 91. The method of embodiment 90, wherein the formulation comprises about 2% to about 3% w / v sucrose.

[0441] 92. The method of embodiment 91, wherein the formulation comprises about 2.5% w / v sucrose.

[0442] 93. The method of any one of embodiments 89-92, wherein the formulation does not comprise one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

[0443] 94. The method of embodiment 93, wherein the formulation does not contain trehalose, MSG, or rHSA.

[0444] 95. The method of any one of embodiments 89 to 94, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

[0445] 96. The method of any one of embodiments 89-95, wherein the Listeria strain is a recombinant Listeria monocytogenes strain, the formulation comprises about 2% to about 3% w / v sucrose, and the formulation does not comprise trehalose, MSG, or rHSA.

[0446] The subject matter disclosed herein also includes, but is not limited to, the following embodiments.

[0447] 1. A method for producing a freeze-dried composition comprising a Listeria strain, comprising: (a) providing a composition comprising a Listeria strain in a formulation comprising a buffer and sucrose; (b) optionally cooling the composition provided in step (a) in a freezing step, where the temperature is between about -32°C and -80°C; (c) optionally exposing the composition produced by step (b) to a vacuum in a primary drying step, where the temperature is between about -10°C and -30°C; and (d) optionally exposing the composition produced by step (c) to a vacuum in a secondary drying step, where the temperature is between about 5°C and 25°C, where the temperature is between about 5°C and 20°C, thereby producing a freeze-dried composition.

[0448] 2. The method of embodiment 1, wherein prior to step (a), a stress response is induced in the Listeria strain by exposing the Listeria strain to a reduced temperature.

[0449] 3. The method of embodiment 1, wherein prior to step (a), a stress response is not induced in the Listeria strain by exposing the Listeria strain to a reduced temperature.

[0450] 4. The method of any preceding embodiment, wherein the Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a).

[0451] 5. The method of any one of embodiments 1 to 3, wherein the Listeria strain used in the composition in step (a) is freshly cultured prior to step (a).

[0452] 6. The method of any preceding embodiment, wherein the buffer is a phosphate buffer.

[0453] 7. The method of any preceding embodiment, wherein the formulation comprises 1% to 5% w / v sucrose.

[0454] 8. The method of embodiment 7, wherein the formulation comprises 2% to 3% w / v sucrose.

[0455] 9. The method of any preceding embodiment, wherein the formulation does not include one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

[0456] 10. The method of embodiment 9, wherein the formulation does not contain trehalose, MSG, or rHSA.

[0457] 11. The method of any preceding embodiment, wherein the temperature in the freezing step (b) is between -40°C and -50°C.

[0458] 12. The method of any preceding embodiment, wherein the cooling in the freezing step (b) is for 2 to 4 hours.

[0459] 13. The method of any preceding embodiment, wherein the temperature in the primary drying step (c) is between -12°C and -22°C.

[0460] 14. The method of claim 13, wherein the temperature in the primary drying step (c) is between -17°C and -19°C.

[0461] 15. The method of any preceding embodiment, wherein the primary drying step (c) is for 20 to 30 hours.

[0462] 16. The method of any preceding embodiment, wherein the temperature in the secondary drying step (d) is between 10°C and 20°C.

[0463] 17. The method of any preceding embodiment, wherein the secondary drying step (d) is for 1 to 10 hours.

[0464] 18. The method of embodiment 17, wherein the secondary drying step (d) is for 1 to 3 hours.

[0465] 19. The method of any preceding embodiment, wherein the residual moisture in the lyophilized composition is between 1% and 5%.

[0466] 20. The method of embodiment 19, wherein the residual moisture in the lyophilized composition is between 3% and 4%.

[0467] 21. The method of any preceding embodiment, wherein the lyophilized composition exhibits at least 60% viability after storage at -20°C or 4°C for 6 months.

[0468] 22. The method of embodiment 21, wherein the freeze-dried composition exhibits a viability of at least 75% after storage at -20°C or 4°C for 6 months.

[0469] 23. The method of embodiment 22, wherein the lyophilized composition exhibits at least 80% viability after storage at -20°C or 4°C for 6 months.

[0470] 24. The method of any preceding embodiment, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

[0471] 25. The Listeria strain is a recombinant Listeria monocytogenes strain, the buffer is a phosphate buffer, and the formulation is 2% to 3% w / v sucrose, and the formulation does not contain trehalose, MSG, or rHSA; the holding temperature in the primary drying step (c) is between -17°C and -19°C; the temperature in the secondary drying step (d) is between -10°C and 20°C; and the residual moisture in the lyophilized composition is between 3% and 4%.

[0472] 26. The method of any preceding embodiment, wherein the Listeria strain is a recombinant Listeria strain comprising a nucleic acid including a first open reading frame encoding a fusion polypeptide, and the fusion polypeptide comprises a PEST-containing peptide fused to a disease-associated antigenic peptide.

[0473] 27. The method of embodiment 26, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion or inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

[0474] 28. The method of embodiment 26, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising deletions or inactivating mutations in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO.

[0475] 29. A formulation for lyophilization of a Listeria strain, comprising: (1) a Listeria strain; (2) a phosphate buffer; and (3) sucrose.

[0476] 30. The formulation of embodiment 29, wherein the Listeri...

Claims

1. 1. A formulation for lyophilization comprising: (1) a Listeria strain; (2) a phosphate buffer; and (3) sucrose, the Listeria strain is a recombinant Listeria monocytogenes strain; the formulation comprises 2% to 3% w / v sucrose; The formulation does not contain trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA); formulation.

2. 2. The formulation of claim 1, wherein the Listeria strain is a strain in which a stress response has been induced by exposing the Listeria strain to a temperature between 0°C and 4°C.

3. 3. The formulation of claim 1 or 2, wherein the Listeria strain is a Listeria strain obtained from a frozen Listeria stock.

4. 3. The formulation of claim 1 or 2, wherein the Listeria strain is a Listeria strain obtained from a freshly cultured Listeria stock.

5. A formulation according to any one of claims 1 to 4 comprising 2.5% w / v sucrose.

6. 6. The formulation of any one of claims 1-5, wherein the recombinant Listeria monocytogenes strain comprises a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, the fusion polypeptide comprising a PEST-containing peptide fused to a disease-associated antigenic peptide.

7. 7. The formulation of claim 6, wherein the recombinant Listeria monocytogenes strain is an attenuated Listeria monocytogenes strain comprising a deletion of prfA or an inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein.

8. 7. The formulation of claim 6, wherein the recombinant Listeria monocytogenes strain is an attenuated Listeria monocytogenes strain comprising deletions of actA, dal and dat or inactivating mutations in actA, dal and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO.

9. 1. A lyophilized composition comprising a Listeria strain, a phosphate buffer, and sucrose, the Listeria strain is a recombinant Listeria monocytogenes strain; The lyophilized composition does not contain trehalose, monosodium glutamate (MSG), or recombinant human serum albumin (rHSA); the residual moisture in the lyophilized composition is between 1% and 5%; the recombinant Listeria monocytogenes strain comprises a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, the fusion polypeptide comprising a PEST-containing peptide fused to a disease-associated antigenic peptide; (I) the recombinant Listeria monocytogenes strain is an attenuated Listeria monocytogenes strain comprising a deletion of prfA or an inactivating mutation in prfA, and the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein; or (II) the recombinant Listeria monocytogenes strain is an attenuated Listeria monocytogenes strain comprising a deletion of actA, dal, and dat or an inactivating mutation in actA, dal, and dat, the nucleic acid is on an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and the PEST-containing peptide is an N-terminal fragment of LLO. Lyophilized composition.

10. 10. The lyophilized composition of claim 9, wherein the residual moisture in the lyophilized composition is between 2% and 4%.

11. 11. The freeze-dried composition of claim 9 or 10, wherein the residual moisture in the freeze-dried composition is at least 2.5%.

12. 12. The lyophilized composition of claim 11, wherein the residual moisture in the lyophilized composition is at least 3%.

13. The lyophilized composition of any one of claims 9 to 12, exhibiting at least 60% viability after storage between -20°C and 4°C for 12 months.

14. 14. The lyophilized composition of claim 13, exhibiting at least 75% viability after storage between -20°C and 4°C for 12 months.

15. 15. The lyophilized composition of claim 14, exhibiting at least 80% viability after storage between -20°C and 4°C for 12 months.

Images