Composition and method for lyophilization of bacterium or listeria strain

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 on September 19, 2017, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Reference to a Sequence Listing Submitted as a Text File via EFS - Web The sequence listing described in the file 519152SEQLIST.txt, which is 89.2 kilobytes in size and was created on August 24, 2018, is hereby incorporated by reference in its entirety.

Background Art

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

[0004] Compared with liquid formulations, solid formulations have several advantages, such as excellent storage stability, molecular mobility, and reduction of unwanted chemical reactions, as well as lower packaging weight to increase ease of shipping and distribution. Furthermore, since all currently available commercial vaccines require cold storage, the goal is to utilize solid-state stabilization technologies to increase their stability at room temperature or higher temperatures, as well as to reduce dependence on the cold chain for maintaining efficacy and ensuring safety. The low storage and transportation costs of freeze-dried bacterial cultures are a major advantage compared to frozen storage, so freeze-drying is a preferred storage method, but freeze-drying is a very complex physical process that is affected by many parameters that require special equipment and trained personnel. Freeze-drying can cause many types of damage to cells, including a decrease in viability, a decrease in metabolic activity, and a change in cell morphology, which can affect the physiology, characteristics, and functions of cells such as bacteria.

[0005] In addition, the effects of various different freeze-drying parameters are very strain-specific, and due to this strain-dependence, it is difficult to draw general conclusions or guidance from any one particular strain. Due to the high biological and metabolic diversity of bacteria, it is difficult and challenging to develop strain-specific optimized freeze-drying procedures. Data regarding the freeze-drying of Listeria strains such as Listeria monocytogenes, and which parameters need to be optimized to make freeze-drying a viable option for Listeria, and how to optimize those parameters, are very limited. Summary of the Invention Means for Solving the Problems

[0006] Abstract Methods and compositions for the lyophilization of bacteria or Listeria strains, such as Listeria monocytogenes, are provided. In one aspect, a method for producing a lyophilized composition containing bacteria or a Listeria strain is provided. Some such methods can include providing a composition containing bacteria or a Listeria strain in a formulation containing 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 secondary drying step, thereby producing a lyophilized composition.

[0007] In some such methods, the bacteria or Listeria strain used in the composition is a frozen Listeria strain that is thawed prior to the freezing step. In certain examples, the frozen bacteria or Listeria strain 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, the thawing is a thawing within about 8 hours. Optionally, the thawed bacteria or Listeria strain is held at a temperature between about 2°C and about 8°C for about 24 hours or less. In certain examples, the concentration of the bacteria or Listeria strain to be thawed can be between about 1×10 9 ~about 1×10 10 colony forming units (CFU) per milliliter.

[0008] In some such methods, the formulation contains a buffer and sucrose. For example, the formulation buffer can contain 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 is about 1×10 per milliliter9 ~about 1×10 10 contains bacteria or Listeria of colony forming units (CFU).

[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 shows a survival rate 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 a method can include, for example, (a) providing a composition comprising a Listeria strain in a formulation containing a buffer and sucrose, (b) cooling the composition provided in step (a) to a holding temperature 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 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 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, (a) providing a composition comprising a Listeria strain in a formulation containing a buffer and sucrose, (b) cooling the composition provided in step (a) to a holding temperature 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 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 between about 5°C and about 25°C in a secondary drying step, thereby producing a lyophilized composition. In some such methods, the Listeria strain is a recombinant Listeria monocytogenes strain, exposing the Listeria strain to a reduced temperature induces a stress response in the Listeria strain, the buffer is a phosphate buffer, the formulation contains 2% - 3% w / v sucrose, the formulation does not contain 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 contains about 2% - about 3% w / v sucrose; the formulation does not contain trehalose, MSG, or rHSA; the formulation contains about 1×10 9 ~ about 1×10 10Containing Listeria of colony forming units (CFU); 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 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 thawed prior to step (a). Optionally, such methods have one or more or all of the following elements: the concentration of the frozen Listeria strain to be thawed is about 1×10 9 ~ about 1×10 10 colony forming units (CFU); the frozen Listeria strain is thawed at about 37°C; the frozen Listeria strain is thawed for no more than 8 hours; and the frozen Listeria strain is held at about 2°C to about 8°C for no more than 24 hours after thawing. Also provided are freeze-dried bacteria or Listeria strains produced by the freeze-drying methods disclosed herein.

[0014] In another aspect, a formulation for freeze-drying containing bacteria or a Listeria strain is provided. Such a formulation can 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 aspect, a lyophilized composition comprising a bacterium or a Listeria strain is provided. Some such lyophilized compositions have at least about 2.5% or at least about 3% residual moisture. 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 in the lyophilized composition is between 3% and 4%.

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

Brief Description of the Drawings

[0017]

Figure 1

[0018]

Figure 2A

Figure 2B

[0019]

Figure 3

[0020]

Figure 4

[0021]

Figure 5

[0022]

Figure 6

[0023]

Figure 7A

Figure 7B

[0024]

Figure 8

[0025]

Figure 9

[0026]

Figure 10

[0027]

Figure 11

[0028]

Figure 12

[0029]

Figure 13

[0030]

Figure 14

[0031]

Figure 15

[0032]

Figure 16

[0033]

Figure 17

[0034]

Figure 18

[0035]

Figure 19

[0036]

Figure 20

[0037]

Figure 21

[0038]

Figure 22A

[0039]

Figure 22B

[0040]

Figure 23A

[0041]

Figure 23B

[0042]

Figure 24A

[0043]

Figure 24B

[0044]

Figure 25

[0045]

Figure 26

[0046]

Figure 27

[0047]

Figure 28

[0048]

Figure 29

[0049]

Figure 30

[0050]

Figure 31

[0051]

Figure 32

[0052]

Figure 33

[0053]

Figure 34

[0054]

Figure 35

[0055]

Figure 36

[0056]

Figure 37

[0057]

Figure 38

[0058]

Figure 39

[0059]

Figure 40

[0060]

Figure 41A

Figure 41B

[0061]

Figure 42

[0062]

Figure 43

[0063]

Figure 44A

Figure 44B

[0064]

Figure 45A

Figure 45B

[0065]

Figure 46

[0066]

Figure 47

[0067]

Figure 48

[0068]

Figure 49

[0069]

Figure 50

[0070]

Figure 51

[0071]

Figure 52

[0072]

Figure 53

[0073]

Figure 54

[0074]

Figure 55

[0075]

Figure 56

[0076]

Figure 57

[0077]

Figure 58A

Figure 58B

[0078]

Figure 58C

Figure 58D

[0079]

Figure 59A

Figure 59B

[0080]

Figure 59C

Figure 59D

[0081]

Figure 60A

Figure 60B

[0082]

Figure 60C

Figure 60D

[0083]

Figure 61

[0084]

Figure 62

[0085]

Figure 63A

[0086]

Figure 63B

[0087]

Figure 64

[0088]

Figure 65

[0089] Definitions As used herein, the terms "protein", "polypeptide" and "peptide" in the same sense refer to amino acids in polymeric form of any length, including encoded and non-encoded amino acids, as well as chemically or biochemically modified or derivatized amino acids. These terms include modified polymers such as polypeptides having a modified peptide backbone.

[0090] A protein is said to have an "N-terminus" and a "C-terminus". The term "N-terminus" relates to the starting point of a protein or polypeptide having an amino acid with a free amine group (-NH2) at the terminus. The term "C-terminus" relates to the end point of an amino acid chain (protein or polypeptide) having a free carboxyl group (-COOH) at the terminus.

[0091] The term "fusion protein" refers to a protein that contains two or more peptides linked to each other by peptide bonds 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 can be recombinantly expressed as a single-chain fusion protein. Alternatively, the peptides may be linked to each other by a "linker" between two or more peptides, for example, one or more amino acids or another suitable linker.

[0092] The terms "nucleic acid" and "polynucleotide" as used herein in the same sense refer to nucleotides in polymeric form of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-stranded, double-stranded 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, unnatural or derivatized nucleotide bases.

[0093] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to form oligonucleotides in such a way that the 5' phosphate ester of one mononucleotide pentose ring is joined in one direction by a phosphodiester linkage to the 3' oxygen of the adjacent one. The end of an oligonucleotide is called the "5' end" if its 5' phosphate ester is not linked to the 3' oxygen of a mononucleotide pentose ring. The end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate ester of another mononucleotide pentose ring. A nucleic acid sequence may also be said to have 5' and 3' ends even if it is within a larger oligonucleotide. In either a linear or circular DNA molecule, a distinct element is said to be "downstream" or 3' of an "upstream" or 5' element.

[0094] "Codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one codon of a native sequence with a codon that is more frequently used or most frequently used in the genes of a host cell 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 codons with higher usage frequencies as compared to nucleic acid sequences that naturally occur in a given Listeria cell or any other host cell. Codon usage tables are readily available, for example, in the "Codon Usage Database". The optimal codons utilized by L. monocytogenes for each amino acid are shown in US2007 / 0207170, which is hereby incorporated by reference in its entirety for all purposes. These tables can be adapted in a number of ways. Nakamura et al. (2000) Nucleic Acids Research, which is hereby incorporated by reference in its entirety for all purposes, See 28:292. Computer algorithms for codon optimization of specific sequences for expression in a particular host are also available (see, for example, Gene Forge).

[0095] The terms "plasmid" or "vector" include any known delivery vector, including bacterial delivery vectors, viral vector delivery vectors, delivery vectors for peptide immunotherapy, delivery vectors for DNA immunotherapy, episomal plasmids, integrating plasmids, or phage vectors. The term "vector" means a construct that can deliver one or more fusion polypeptides into a host cell and, optionally, cause expression in the 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., it is episomal or extrachromosomal and not integrated into the genome of the host cell), and that replicates independently of chromosomal DNA. A plasmid can be linear, or circular, single-stranded, or double-stranded. An episomal plasmid can, if desired, persist in multiple copies in the cytoplasm of a host cell (e.g., Listeria), resulting in amplification of any desired gene within the episomal plasmid.

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

[0098] The term "stably maintained" refers to the 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 generations, 20 generations, at least 25 generations, at least 30 generations, at least 40 generations, at least 50 generations, at least 60 generations, at least 80 generations, at least 100 generations, at least 150 generations, at least 200 generations, at least 300 generations, or at least 500 generations. "Stably maintained" can refer to a nucleic acid molecule or plasmid that is stably maintained intracellularly (e.g., in culture) in vitro, stably maintained in vivo, or both.

[0099] An "open reading frame" or "ORF" is a portion of DNA that contains a sequence of bases that could potentially encode 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, usually containing a TATA box, that can direct RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site of a specific 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 operably linked polynucleotides. A promoter can 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). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporal-specific promoter (e.g., a developmental regulatory 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, which is hereby incorporated by reference in its entirety.

[0101] "Operably linked" or "operably connected" refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function properly and at least one of the components allows the possibility of mediating the function exerted on at least one of the other components. For example, if a promoter controls the transcription level of a coding sequence in response to the presence or absence of one or more transcriptional regulatory factors, the promoter can be operably linked to the coding sequence. Operable linkage can include such sequences that are contiguous to each other or act in trans (e.g., a regulatory sequence can act to control the transcription of a coding sequence from some distance away).

[0102] With respect to two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to residues in the two sequences that are the same when aligned to maximize matches over a specified comparison window. When percentage of sequence identity is used in reference to a protein, positions of non-identical residues are often recognized to differ by conservative amino acid substitutions where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus, does not change the functional properties of the molecule. When sequences differ by conservative substitutions, the percentage of sequence identity can be adjusted upwards 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 of sequence identity by scoring conservative substitutions as partial matches rather than complete mismatches. Thus, for example, if a score of 1 is assigned to identical amino acids and a score of 0 is assigned to non-conservative substitutions, a score between 0 and 1 is assigned to conservative substitutions. Scoring of conservative substitutions is calculated, for example, according to the implementation of the program PC / GENE (Intelligenetics, Mountain View, California).

[0103] "Percentage of sequence identity" is a value determined by comparing two sequences optimally aligned over a comparison window (the maximum number of exact match residues), wherein a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences (which does not include additions or deletions). This percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of match positions, dividing the number of match positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes concatenated heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0104] Unless otherwise specified, array identity / similarity values refer to values obtained using GAP Version 10 with the following parameters: % 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 program equivalent thereto. An "equivalent program" includes any sequence comparison program that, for any two sequences in question, generates an alignment having the same nucleotide or amino acid residue matches and the same percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

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

[0106]

Table 1

[0107] An "identical" sequence (e.g., a nucleic acid sequence) refers to a sequence that is identical or substantially similar to a known reference sequence, such that it is, for example, 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 seen in a normal state or situation (as contrasted with a state or situation that is mutated, diseased, altered, etc.). Wild type genes and polypeptides often exist in multiple different 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 includes those that are up to a substantially pure preparation of the protein and polynucleotide. The term "isolated" also includes proteins and nucleic acids that do not have a corresponding non-naturally occurring counterpart, are chemically synthesized and thus are not substantially contaminated by other proteins or nucleic acids, or are separated or purified from most of the other cellular components that naturally accompany them (e.g., other cellular proteins, polynucleotides or cellular components).

[0110] An "exogenous" or "heterologous" molecule or sequence is a molecule or sequence that is not normally expressed in a cell or 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, an exogenous or heterologous molecule or sequence may include a mutant version of the corresponding endogenous sequence within the cell, or a sequence corresponding to an endogenous sequence that is within the cell but in a different form (i.e., not within the chromosome). An exogenous or heterologous molecule or sequence in a particular cell may also include a molecule or sequence derived from a species different from the reference species of that cell or from a different organism within the same species. For example, in the case of a Listeria strain expressing a heterologous polypeptide, the heterologous polypeptide is 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 derived from a different organism within the same species.

[0111] In contrast, an "endogenous" molecule or sequence or a "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 a population but is still similar enough in a 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 compared to another isoform, or version (e.g., of the same protein). For example, a protein isoform may be produced from related but different genes, may result from alternative splicing of the same gene, or may result from a single nucleotide polymorphism.

[0114] The term "fragment" when referring to a protein means a protein that is shorter than the full-length protein, or a protein having 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 a nucleic acid having 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 a naturally occurring protein by conservative amino acid differences, by modifications that do not affect the amino acid sequence, or by both.

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

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

[0118] As used herein, the term "antigen" refers 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. An antigen can be, for example, a lipid, protein, carbohydrate, nucleic acid, or a combination and variant thereof. For example, an "antigenic peptide" refers to a peptide that, when present in or detected by a subject or organism, results in an increase in the immune response in the subject or organism. For example, such an "antigenic peptide" can be a protein that is 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, and recognition or detection of which results in an increase in the immune response against that 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., the site to which an antibody binds). Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are usually retained even when exposed to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are usually lost when treated with denaturing solvents. Epitopes generally contain at least 3, more usually at least 5 or 8 - 10 amino acids in a specific 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. 66, Glenn E. Morris, Ed. (1996), which is hereby incorporated by reference in its entirety for all purposes.

[0120] The term "mutation" refers to any change in the structure of a gene or protein. For example, a mutation can occur as a result of a deletion, insertion, substitution, or rearrangement of a chromosome or protein. "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. "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 "frameshift" mutation in DNA occurs when an addition or loss of nucleotides changes the reading frame of a gene. The reading frame consists of groups of three bases, each of which codes for one amino acid. A frameshift mutation shifts the grouping of these bases, changing the amino acid code. The resulting protein is usually non-functional. Insertions and deletions are each frameshift mutations.

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

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

[0124] The term "in vitro" refers to an artificial environment and to processes or reactions that occur in an artificial environment (e.g., a test tube).

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

[0126] The term "frozen state glass transition temperature" (Tg’) refers to the following. When heated, a solution of a sugar glass undergoes a secondary 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 the following. Similar 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 highest temperature at which a product can withstand the primary drying without losing its physical structure.

[0129] The term "drug substance" (DS) refers to the active ingredient. The drug substance refers to any component of a drug product that is intended to provide a 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 body of a human or other animal. The active ingredient includes the components of the product that can undergo chemical changes during the manufacture of the drug product and can be present in the drug product in a modified form for the purpose of providing the designated 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 becomes the active ingredient or the finished dosage form of the drug when used in the manufacture, processing or packaging of the drug, but this term does not include intermediates used in the synthesis of such substances.

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

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

[0133] The specification of a range of values includes all integers within the range or defining the range, and all sub-ranges defined by the integers within the range.

[0134] Unless the context clearly indicates otherwise, the term "about" encompasses values within the standard error of measurement (e.g., SEM) of the stated value or the amount of variation, ±0.5%, 1%, 5%, or 10% 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] Compositions and methods are disclosed herein related to stable lyophilized pharmaceutical formulations prepared by lyophilizing aqueous formulations containing bacteria or Listeria strains, such as Listeria monocytogenes. In some embodiments, the lyophilized formulations are stable for at least 6 months, at least 1 year, or at least 2 years at 4°C or -20°C. In some embodiments, the lyophilized formulations are suitable for parenteral administration, such as intravenous injection.

[0138] For example, frozen liquid formulations currently used in therapeutics containing L. monocytogenes are stored and shipped at -80°C. This low temperature presents supply chain challenges, especially in countries in South America and Africa, both for shipping of materials and storage at the clinical site. Therefore, it is desirable to have a refrigerated or -20°C supply chain. Optimization of the manufacturing processes described herein makes it possible to produce stable drug products that can be maintained at higher temperatures. Counterintuitively, the examples described herein show that higher residual moisture (e.g., higher than normal 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., sufficiently higher than Tg, about -17°C to about -19°C or about -18°C in one embodiment) improved the stability of the lyophilized product. In addition, preconditioning by heat shock of the cells prior to lyophilization improved the stability of the lyophilized product. In addition, the use of higher viable bacterial concentrations (viable cell counts, or VCC) results in improved stability of the lyophilized drug product compared to lower VCC. In addition, counterintuitively, thawing of the frozen drug substance at about 37°C prior to lyophilization improved the stability of the lyophilized drug product compared to thawing at room temperature or 2 - 8°C. These process improvements improve stability at higher temperatures compared to liquid frozen formulations, resulting in a higher temperature supply chain. This enables 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., a target residual moisture level higher than the normal target residual moisture level, which can be achieved by changing the secondary drying temperature and, if necessary, the secondary drying time, for example, the use of about 3.5% in one embodiment), the process of inducing a stress response within L. monocytogenes cells by a temperature shift prior to lyophilization improves the stability at higher temperatures for the lyophilized drug product. Similarly, the use of a formulation containing a phosphate buffer and a lower level of sucrose than normal levels (e.g., about 2.5% w / v sucrose), and the use of a high primary drying step temperature (e.g., about -18 °C in one embodiment) improve the stability at higher temperatures for the lyophilized drug product, 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 the stability at higher temperatures compared to liquid frozen formulations, and as a result, enable a higher temperature supply chain. This enables an easier-to-handle supply chain and enables distribution to countries where storage at -80 °C is not feasible. II. Freeze-drying of bacteria or Listeria

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

[0141] Lyophilization involves manipulating the temperature and pressure of a solution such that the solvent phase transitions directly from the frozen state to the gaseous state without passing through the liquid phase / liquid state. This is achieved by cooling the solution and reducing the pressure below the triple point of water. This enables the removal of the solvent from the product without subjecting the product to high heat. During the freezing stage, the formulation is cooled. As pure crystalline ice forms from the liquid, the remainder of the liquid is cryoconcentrated into a more viscous state, thereby inhibiting further crystallization. Eventually, this highly concentrated and viscous solution solidifies, resulting in an amorphous phase, a crystalline phase, or a composite amorphous-crystalline phase. During the primary drying stage, the ice formed during freezing is removed by sublimation at a temperature below ambient temperature under vacuum. Throughout this stage, the product is maintained in a solid state below the collapse temperature of the product to dry the product while retaining the structure established in the freezing step. This collapse temperature is the glass transition temperature (Tg’) for amorphous products or the eutectic temperature (Te) for crystalline products. During the secondary drying stage, a relatively small amount of bound water remaining in the matrix is removed by desorption. During this stage, the temperature of the shelves and the product is increased to facilitate a proper desorption rate and to achieve the desired residual moisture.

[0142] The target profile of a lyophilized drug product is a profile that results in a well-defined cake that is stable either at 2 - 8 °C or -20 °C and retains the same potency and biological activity as the liquid frozen formulation at the target residual moisture. Protective strategies that can improve bacterial viability during freeze-drying include, for example, adding excipients to the drying medium, controlling process parameters, prestressing the bacterial sample prior to freeze-drying, and varying the fermentation conditions of the bacteria. However, the efficiency of these strategies is strain-dependent. This is because the inherent tolerance to the drying process also varies by strain. Even very closely related bacterial strains may have one strain that is far more resistant to the freeze-drying process than another. Due to this strain-dependence, it is difficult to draw general conclusions or guidance.

[0143] Methods for producing freeze-dried compositions comprising bacteria or Listeria strains are provided herein. Such methods can include providing a composition comprising bacteria 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 secondary drying step, thereby producing a freeze-dried composition.

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

[0145] In some such methods, the formulation comprises a buffer and sucrose. For example, the formulation buffer can 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 is from about 1×10 9 ~about 1×1010 It contains bacteria or Listeria in colony forming units (CFU).

[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 survival rate 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: (a) providing a composition containing bacteria or a Listeria strain in a formulation containing a buffer and sucrose; (b) cooling the composition provided in step (a) at a holding temperature 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 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 between about -5°C and about 25°C in a secondary drying step.

[0151] Further embodiments for cell preconditioning, formulation, freezing step, primary drying step, secondary drying step, and the lyophilized product are provided below. A. Preconditioning of Bacteria or Listeria

[0152] The cultures of bacteria or Listeria strains used in the lyophilization method disclosed herein can be from a frozen stock, from a starter culture, or from colonies (e.g., freshly cultured bacteria or Listeria).

[0153] Provided herein is a method for preparing a frozen bacterium or Listeria strain for lyophilization, the method comprising the step of thawing the frozen bacterium or Listeria strain. When the bacterium or Listeria strain is from a frozen stock, it can be thawed by any means. The temperature and time for thawing can affect stability. By identifying the appropriate conditions for thawing the frozen drug substance, it becomes possible to freeze and hold the drug substance before lyophilization. By ensuring that the resulting cells from thawing are of high quality and healthy, it is ensured 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 can be incubated, for example, for about 0.5, 1, 2, 3, 4 hours or longer. In another example, it can be thawed at about 37°C and can be incubated, for example, for about 0.5, 1, 2, 3, 4 hours or longer.

[0154] In one example, the frozen bacterium or Listeria strain 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 at about 37°C.

[0155] The frozen bacteria or Listeria strains can be thawed, for example, for about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours, between about 0.5 to about 8 hours, between about 1 to about 8 hours, between about 2 to about 8 hours, between about 3 to about 8 hours, between about 4 to about 8 hours, between about 5 to about 8 hours, between about 6 to about 8 hours, or between about 7 to about 8 hours. Alternatively, the frozen bacteria or Listeria strains can be thawed, for example, for less than about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0156] The frozen bacteria or Listeria strains to be thawed may be in a bacterial or Listeria lyophilized formulation, or may be thawed in a bacterial or Listeria lyophilized formulation. Such bacterial or Listeria lyophilized formulations are disclosed in more detail elsewhere in this specification.

[0157] The frozen bacteria or Listeria strains can be held at a certain temperature after thawing. For example, the frozen bacteria or Listeria strains can be held at a temperature between about 2°C to about 8°C after thawing. The frozen bacteria or Listeria strains can be held, for example, for 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 between about 0.5 to about 24 hours, between about 1 to about 24 hours, between about 2 to about 24 hours, between about 5 to about 24 hours, between about 10 to about 24 hours, between about 12 to about 24 hours. Alternatively, the frozen bacteria or Listeria strains can be held, for example, for less than 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. In certain examples, the frozen bacteria or Listeria strains are thawed at a temperature of about 37°C for less than about 8 hours and held at a temperature between about 2°C to about 8°C for less than about 24 hours.

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

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

[0160] The nutrient medium utilized to grow the bacteria or Listeria strain can be any suitable nutrient medium. Examples of suitable media include, for example, Luria broth (LB: Luria-Bertani broth); Terrific broth (TB); modified Terrific broth without animal substances; or minimal medium. The bacteria or Listeria strain can be cultured by any known means for growing cells. For example, the growth step can be carried out using a shaking flask (e.g., a baffled shaking flask), a batch fermenter, a stirred tank or flask, an airlift fermenter, a fed-batch, a continuous cell reactor, a cell immobilization reactor, or any other means for bacterial growth.

[0161] Optionally, a constant pH is maintained during the growth of the culture (e.g., in a batch fermenter). 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, between about 6.5 and about 7.5, between about 6.0 and about 8.0, between about 6.0 and about 7.0, between about 6.0 and about 7.0, or between about 6.5 and about 7.5. Alternatively, immediately after collecting the cells from the bioreactor, the pH can be decreased by adding an acid to induce a stress response, and this stress response can activate a series of genes that can better prepare the cells for lyophilization.

[0162] Optionally, a constant temperature can be maintained during the 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 collecting the cells from the bioreactor, the temperature can be decreased by placing the cells in an ice bath (e.g., about 0°C or about 4°C) to induce a stress response, which can activate a set of genes that can better prepare the cells for lyophilization.

[0163] Optionally, a constant dissolved oxygen concentration can be maintained during the 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 nearly 100% saturation.

[0164] The bacteria or Listeria strain can be passaged through an animal host prior to lyophilization, if desired. Such passaging 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, remove unstable substrains of the Listeria strain, or prevent the spread of unstable substrains of the Listeria strain. Methods for passaging Listeria strains through an animal host are well known and are described, for example, in US2006 / 0233835, which is hereby incorporated by reference in its entirety for all purposes. B. Bacterial or Listeria Lyophilized Formulation

[0165] Prior to lyophilization, bacteria or Listeria strains can be supplied to a suspension (formulation) containing a buffer and an excipient. The design of the formulation to be lyophilized can depend on the pharmaceutical active ingredient and the requirements of 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 regulators (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 certain examples, 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 in this specification, suitable survival levels of Listeria monocytogenes are achieved using phosphate buffers. In some such buffers, the concentration of KH 2 PO 4 (anhydrous) is between about 0.1 and 0.3 g / L, 0.12 and 0.28 g / L, 0.14 and 0.26 g / L, 0.16 and 0.24 g / L, 0.18 and 0.22 g / L, 0.19 and 0.21 g / L, or 0.2 g / L. In some such buffers, Na 2 HPO 4The concentration of (anhydrous) is between about 1.0 and 1.3 g / L, 1.02 to 1.28 g / L, 1.04 to 1.26 g / L, 1.06 to 1.24 g / L, 1.08 to 1.22 g / L, 1.1 to 1.2 g / L, 1.12 to 1.18 g / L, 1.14 to 1.16 g / L, or 1.15 g / L. Some such buffers are about 5 to 20, 6 to 18, 7 to 16, 8 to 14, 9 to 12, 9 to 11, or 10 mM. Some such buffers have a pH of about 6.8 to 7.6, 6.9 to 7.5, 7.0 to 7.4, 7.1 to 7.3, or 7.2. As an example, a phosphate buffer can have between about 0.19 and 0.21 g / L (e.g., 0.2 g / L) of KH 2 PO 4 (anhydrous), between about 1.14 and 1.16 g / L (e.g., 1.15 g / L) of Na 2 HPO 4 (anhydrous) and can have a pH of about 7.1 to 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 lyophilization process. Cryoprotectants are water-soluble chemical substances that lower the melting point of water. When ice crystals form, bacterial cells are compressed into the unfrozen fraction. The addition of cryoprotectants can expand the unfrozen section and provide a larger space for bacterial cells, which can lead to a reduction in cell damage caused by mechanical stress 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 skim 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 a specific example, the excipient contains, consists essentially of, or consists 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 can 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, skim milk, glucose, maltose, lactose, mannitol, glycine, glycerol, sodium chloride, yeast extract, dextran, dextrose, polydextrose, monosodium glutamate, maltodextrin, ascorbic acid, saccharides 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 between about 1×10 9 ~ about 1×10 10 colony forming units (CFU) per milliliter. C. Freezing step

[0170] The first step in lyophilization is the freezing step. During this stage, the formulation is cooled. This can be achieved, for example, by lowering the temperature of the shelves of the lyophilizer in a shelf freeze dryer (i.e., by lowering the shelf temperature). Ice crystals are formed during freezing, which can damage bacteria. The growth of ice crystals 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, and thus can reduce cell damage. The formation of ice crystals can be harmful to bacteria. When water crystallizes, the solutes in the remaining unfrozen fraction become concentrated, 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 always coincide with the best survival rate results. The optimal freezing conditions can vary depending on the cryoprotectant used in the formulation and the bacterial strain.

[0171] The temperature (e.g., shelf temperature) can reach the holding temperature (e.g., shelf temperature) of the freezing step by decreasing it at a rate of, for example, about 0.2°C to about 2.0°C per minute. Alternatively, the temperature (e.g., shelf temperature) can reach the holding temperature (e.g., shelf temperature) of the freezing step by decreasing it 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 decreased 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 specific example, the temperature is decreased to the holding temperature at a rate of about 1°C per minute to reach the holding temperature of the freezing step.

[0172] The freezing step can be any suitable time step for freezing bacteria or Listeria strains. Similarly, the temperature can be maintained at the freezing temperature for any suitable time for freezing bacteria or Listeria strains. For example, the freezing step can be a step of 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 maintained at the freezing temperature for the aforementioned time. In a specific example, the freezing step can be a 3.5-hour step, or the temperature can be maintained 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 maintained 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 maintained at the freezing temperature for 1.5 hours. In another specific example, the total time of the freezing step (e.g., raising the temperature to the freezing temperature and then maintaining it at the holding temperature) is about 3.5 to 4.5 hours or about 3.5 hours.

[0173] The freezing temperature (i.e., the holding temperature) can be any temperature suitable for freezing the bacteria or Listeria strain. In some embodiments, the freezing temperature (e.g., the shelf temperature) is a freezing temperature such that the temperature of the formulation is below the glass transition temperature of the solution, and in the case of a sucrose formulation, it can be, for example, about -32°C. Temperatures higher than this temperature cannot truly freeze the solution, and in that case, the solution may collapse during lyophilization, thus potentially leading to a decrease in viability. For example, the temperature (e.g., the 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 specific example, the freezing temperature can be about -45°C. In another example, the temperature can be 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 -33°C and about -75°C, about -33°C and about -70°C, about -33°C and about -65°C, about -33°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 one example, the freezing temperature can be about -39°C. In another example, the freezing temperature can be about -45°C. In a specific example, the holding temperature in the freezing step is between about -40°C and about -50°C (e.g., about -45°C), and the freezing step includes reducing the temperature to the holding temperature at a rate of about 1°C per minute, and the cooling in the freezing step is a cooling of 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 the primary drying step, the composition containing bacteria or Listeria strains produced by the freezing step is exposed to a vacuum at an elevated temperature. In this step, the frozen water is removed by sublimation under vacuum.

[0175] For example, the temperature (e.g., shelf temperature) can be reached to the temperature of the primary drying step by increasing the temperature at a rate of about 0.2 °C to about 2.0 °C per minute. Alternatively, the temperature (e.g., shelf temperature) can be reached to the holding temperature (e.g., shelf temperature) of the primary drying step by increasing the 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 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 specific example, the holding temperature of the primary drying step is reached by increasing the temperature to the holding temperature at a rate of about 1 °C per minute.

[0176] The primary drying step can be for any suitable length of time. Similarly, the holding temperature (e.g., shelf temperature) can be maintained at the primary drying temperature for any suitable length of time. The temperature should be maintained at the primary drying temperature until the primary drying is complete. This time can vary depending on the freeze dryer, vial size, fill volume, number of vials, pressure, and other variable factors. For example, when the product temperature rises to a value at or above the shelf temperature, the end of primary drying can be determined. For example, it can also be determined by a pressure rise test in which the freeze-drying chamber is separated from the vacuum pump and the amount of pressure rising due to continuous sublimation of water is judged. For example, the primary drying step can be a step of 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 maintained at the primary drying temperature for the aforementioned time. In a specific example, the primary drying step can be a step of about 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 hours, or the temperature can be maintained at the primary drying temperature for the aforementioned time. In another specific example, the primary drying step can be a step of about 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 hours, or the temperature can be maintained at the primary drying temperature for the aforementioned time.

[0177] The primary drying step is such that a probe in the freeze dryer (e.g., a probe for the cold spot in the freeze dryer, such as one at the center of the freeze dryer) passes the primary drying holding temperature or T s set point (e.g., about -18 °C), and then it can be a step of a period 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, or the temperature can be maintained at the primary drying holding temperature for the aforementioned period. Alternatively, the drying step is such that the composition to be freeze-dried (e.g., a sample of the composition at the cold spot of the freeze dryer, or all samples of the composition in the freeze dryer) reaches the primary drying holding temperature or T sIt can be a step for a period 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 reaching the set point (e.g., about -18°C), or the temperature can be maintained at the primary drying holding temperature for the aforementioned period. In certain examples, the primary drying step can be, for example, about 30 hours, and a probe in the lyophilizer (e.g., a probe for the cold spot in the lyophilizer such as the one at the center of the lyophilizer) is at the primary drying holding temperature or T s After passing through the set point (e.g., about -18°C) or when the composition to be lyophilized (e.g., a sample of the composition at the cold spot of the lyophilizer or all samples of the composition in the lyophilizer) is at the primary drying holding temperature or T s It can be a step for a period defined as about 14 hours after reaching the set point (e.g., about -18°C), or the temperature can be maintained at the primary drying holding temperature for the aforementioned period.

[0178] The end of the primary drying step is when a probe in the lyophilizer (e.g., a probe for the cold spot in the lyophilizer such as the one at the center of the lyophilizer) is at the primary drying holding temperature or T s It 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 passing through the set point (e.g., about -18°C). Alternatively, the end of the primary drying step is when the composition to be lyophilized (e.g., a sample of the composition at the cold spot of the lyophilizer or all samples of the composition in the lyophilizer) is at the primary drying holding temperature or T s It 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 reaching the set point (e.g., about -18°C). In certain examples, the end of the primary drying step can be, for example, about 30 hours, and a probe in the lyophilizer (e.g., a probe for the cold spot in the lyophilizer such as the one at the center of the lyophilizer) is at the primary drying holding temperature or T sAbout 14 hours after passing through the set point (e.g., about -18 °C), or the composition to be lyophilized (e.g., a sample of the composition at the cold spot of the lyophilizer, or all samples of the composition in the lyophilizer) is at the primary drying hold temperature or T s It can be about 14 hours after reaching the set point (e.g., about -18 °C).

[0179] The primary drying temperature (i.e., the shelf temperature or the holding temperature) can be any suitable temperature for drying bacteria or Listeria strains. For example, the holding 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°, 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°, 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°, 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 certain examples, 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 certain 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 (under vacuum conditions) 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 from about 0.140 to about 0.050, from about 0.100 to about 0.060, from about 0.100 to about 0.070, from about 0.100 to about 0.080, from about 0.099 to about 0.081, from about 0.098 to about 0.082, from about 0.097 to about 0.083, from about 0.096 to about 0.084, from about 0.095 to about 0.085, from about 0.094 to about 0.086, from about 0.093 to about 0.087, from about 0.092 to about 0.088, from about 0.091 to about 0.089, about 0.090 mbar, or about 0.120 mbar. In certain examples, the pressure is about 0.090 mbar.

[0181] In certain examples, the holding temperature in the primary drying step is between about -17 °C and about -19 °C (e.g., about -18 °C), and the primary drying step includes 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 lyophilization is the secondary drying step. In the secondary drying step, the composition containing the bacteria or Listeria strain produced by the primary drying step is exposed to a vacuum at an elevated temperature. In this step, the unfrozen water is removed by desorption.

[0183] For example, the temperature (e.g., shelf temperature) can reach the temperature of the secondary drying step by increasing it at a rate of about 0.2°C to about 2.0°C per minute. Alternatively, the temperature (e.g., shelf temperature) can reach the holding temperature (e.g., shelf temperature) of the secondary drying step by increasing it 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 specific example, the holding temperature of the secondary drying step is reached by increasing the temperature to the holding temperature at a rate of about 0.2°C per minute.

[0184] The secondary drying step can be a step of any suitable duration. Similarly, the temperature (e.g., shelf temperature or holding temperature) can be maintained at the secondary drying temperature for any suitable duration. For example, the temperature can be maintained at the secondary drying temperature for any suitable duration to achieve the desired residual moisture level in the lyophilized product. For example, the secondary drying step can be a step of 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 maintained at the secondary drying temperature for the aforementioned duration. In certain examples, the secondary drying step can be a step of 10 hours or less, 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 maintained at the secondary drying temperature for about 3 hours. In another specific example, the temperature can be maintained 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 or about 6 hours.

[0185] The secondary drying temperature (i.e., the shelf temperature or the holding temperature) can be any temperature suitable for drying bacteria or Listeria strains 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, between about 5°C and about 30°C, between about 10°C and about 30°C, between about 20°C and about 30°C, or between about 15°C and about 25°C. In a specific example, the secondary drying temperature can be about 25°C. In another specific example, the secondary drying temperature can be about 20°C. In another specific example, the secondary drying temperature can be about 20°C or less. In another example, the temperature can be between about 5°C and about 20°C, between about 9°C and about 15°C, between about 10°C and about 15°C, between about 11°C and about 14°C, between 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 specific 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, between about -1°C and about 1°C, or about 0°C. In a specific example, the holding temperature can be between about -5°C and about 5°C or about 0°C.

[0186] The pressure (under vacuum conditions) can be any suitable pressure. In some cases, the pressure is within 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 from 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 specific example, the pressure is about 0.090 mbar.

[0187] In a specific 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 raising 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 to about 6 hours.

[0188] The secondary drying step can obtain a freeze-dried product having any desired residual moisture. For example, the residual moisture can be 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%, 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% or less. Alternatively, the residual moisture can be 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%, 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 about 7% or less.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 can be about 3% and about 4%, about 3.1% and about 3.9%, about 3.2% and about 3.8%, about 3.3% and about 3.7%, about 3.4% and about 3.6%, or about 3.5%. In certain examples, the residual moisture is at least about 2%, at least about 2.5%, or at least about 3%. In another certain example, the residual moisture 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%, 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 lyophilized bacteria or Listeria can be a lyophilized composition comprising any combination of the ingredients listed in the formulation section. In one example, the lyophilized composition comprises a Listeria strain, a buffer (e.g., phosphate), and an excipient (e.g., sucrose). Optionally, the lyophilized composition does not contain one or more or all of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA). Optionally, the lyophilized composition does not contain one or more or all of the ingredients as required, listed in the formulation section.

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

[0191] The lyophilized bacteria can be stored under any suitable conditions, including any suitable temperature, relative humidity, and atmospheric oxygen level, which are well known. The lyophilized bacteria or Listeria can exhibit a survival rate of at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% after reconstitution following storage for a defined period of time. Reconstitution can be performed after storage of the lyophilized 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 of the lyophilized bacteria or Listeria can be, for example, between about 0°C and about 10°C, between about 1°C and about 9°C, between about 2°C and about 8°C, between about 2°C and about 6°C, or between about 3°C and about 5°C. In a specific example, the storage temperature can be between about 2°C and about 8°C, or the storage temperature can be about 4°C. In another example, the storage temperature can be between about -15°C and about -25°C, between about -16°C and about -24°C, between about -17°C and about -23°C, between about -18°C and about -22°C, or between about -19°C and about -21°C. In a specific example, the storage temperature can be about -20°C.

[0193] For example, freeze-dried bacteria or Listeria can exhibit a survival rate 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 can exhibit a survival rate 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. As an example, the freeze-dried bacteria or Listeria can exhibit a survival rate of at least about 75% - about 80% after 6 months at 2 - 8 °C. As another example, the freeze-dried bacteria or Listeria can exhibit a survival rate of at least about 95% - about 100% after 9 months at -20 °C. As another example, the freeze-dried bacteria or Listeria can exhibit a survival rate of at least about 80% - about 90% after 2 months at room temperature or 30 °C. As another example, the freeze-dried bacteria or Listeria can exhibit a survival rate of at least about 60%, 65%, 70%, 75%, 80%, 85%, or 90% after about 12 months, 18 months, or 24 months at about -20 °C. As another example, the freeze-dried bacteria or Listeria can exhibit a survival rate of at least about 60%, 65%, 70%, 75%, 80% after about 12 months, 18 months, or 24 months at about 2 - 8 °C.As another example, lyophilized bacteria or Listeria can exhibit a survival rate of at least about 60%, 65%, 70%, 75% or 80% after about 12 months, 18 months or 24 months at about 2 - 8°C.

[0194] After storage, the lyophilized bacteria or Listeria strain can be reconstituted with a solvent or diluent (e.g., water) as needed. As an example, the solvent or diluent can be a suitable medium for culturing the bacteria or Listeria strain. Methods for reconstitution and rehydration of lyophilized bacteria or Listeria strains are well known. In one example, the volume of the solvent used is the volume of the pre - lyophilization solution used to produce the lyophilized bacteria or Listeria strain. In another example, the volume of the solvent used is more than the volume of the pre - lyophilization solution used to produce the lyophilized bacteria or Listeria strain. In another example, the volume of the solvent used is less than the volume of the pre - lyophilization solution used to produce the lyophilized bacteria or Listeria strain.

[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 have undergone the lyophilization methods disclosed herein, contain bacterial strains, such as Listeria strains. Such bacterial strains may be recombinant bacterial strains. Such recombinant bacterial strains may contain the recombinant fusion polypeptides disclosed herein, or may contain nucleic acids encoding recombinant fusion polypeptides as disclosed elsewhere herein. In some embodiments, the bacterial strain is a Listeria strain, such as a Listeria monocytogenes (Lm) strain. Lm has a number of unique advantages as a vaccine vector. This bacterium grows very efficiently in vitro without special requirements, and this bacterium lacks LPS, a major virulence factor in Gram-negative bacteria such as Salmonella. Genetically attenuated Lm vectors can be easily eliminated with antibiotics if severe adverse effects occur, and unlike some viral vectors, they do not cause integration of genetic material into the host genome, providing additional safety.

[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 the following: 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); L. monocytogenes DP-L4056 that has been phage-cured (see, e.g., Lauer et al. (2002) J Bact 184:4177-4186); L. monocytogenes DP-L4027 that has been phage-cured and has 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 that 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 See Biol 150:527-538); L. monocytogenes DP-L4042 (delta PEST) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci. USA 101:13832-13837 and Supporting Information); L. monocytogenes DP-L4097 (LLO-S44A) (e.g., see 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) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and Supporting Information); L. monocytogenes DP-L4405 (delta inlA) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and Supporting Information); L. monocytogenes DP-L4406 (delta inlB) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and Supporting Information); L. monocytogenes CS-LOOOl (delta actA; delta inlB) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and Supporting Information); L. monocytogenes CS-L0002 (delta actA; delta lplA) (e.g., see Brockstedt et al. (2004) Proc Natl Acad Sci See 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 See 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 having a deletion of lplA1 (encoding lipoic acid protein ligase LplA1) (see, e.g., O’Riordan et al. (2003) Science 302:462-464); L. monocytogenes DP-L4017 (10403S having LLO L461T) (see, e.g., US7,691,393); L. monocytogenes EGD (see, e.g., GenBank accession number AL591824). In another embodiment, the Listeria strain is L. monocytogenes EGD-e (see, e.g., GenBank accession number NC_003210; ATCC accession number BAA-679); L. monocytogenes DP-L4029 (actA deletion, optionally with uvrAB deletion) (DP-L4029uvrAB) (see, e.g., US7,691,393); L. monocytogenes actA- / inlB-double mutant (see, e.g., ATCC accession number PTA-5562); L. monocytogenes lplA mutant or hly mutant (see, e.g., US2004 / 0013690); L. monocytogenes dal / dat double mutant (see, e.g., US2005 / 0048081).Other L. monocytogenes strains include nucleic acids 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 the growth, spread, degradation of single - layer vesicles, degradation of double - layer vesicles, binding to host cells, or uptake by host cells (e.g., by plasmid and / or by genomic integration). Each of the above references is hereby incorporated by reference in its entirety for all purposes.

[0198] Recombinant bacteria or Listeria may have wild - type virulence, attenuated virulence, or no virulence. For example, recombinant Listeria may have sufficient virulence to escape from phagosomes or phagolysosomes and enter the cytosol. Such Listeria strains may be live - attenuated Listeria strains that contain at least one attenuation mutation, deletion, or inactivation disclosed elsewhere in this specification. In some embodiments, recombinant Listeria is an attenuated auxotrophic strain. Auxotrophic strains are those that cannot synthesize specific organic compounds required for their growth. Examples of such strains are described in US8,114,414, which is hereby incorporated by reference in its entirety 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 of the recombinant Listeria strains provided herein contain a plasmid that includes 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. A bacterium or Listeria strain comprising a recombinant fusion polypeptide or comprising a nucleic acid encoding a recombinant fusion polypeptide

[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 a bacterium or Listeria strain comprising a nucleic acid encoding a recombinant fusion protein, the nucleic acid can be codon-optimized. Examples of optimal codons utilized by L. monocytogenes for each amino acid are shown in US2007 / 0207170, which is hereby incorporated by reference in its entirety for all purposes. A nucleic acid is codon-optimized if at least one codon in the nucleic acid is replaced with a codon that has a higher frequency of use by L. monocytogenes for that amino acid compared to the codon in the original sequence.

[0202] The nucleic acid may be present in an episomal plasmid in a bacterium or Listeria strain, and / or the nucleic acid 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 in which the nucleic acid is in an episomal plasmid and one integrated into the genome of the bacterium or Listeria strain.

[0203] The episomal plasmid can be stably maintained in vitro (in cell culture), in vivo (in a host), or both in vitro and in vivo. In the case of 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 a foreign promoter / regulatory sequence or may be operably linked to an endogenous promoter / regulatory sequence. Examples of promoter / regulatory sequences useful for driving constitutive expression of a gene 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 subject to regulatory constraints that often result from integration into genomic DNA and is also not fragmented, and in some cases, the presence of the inserted heterologous gene does not result in rearrangement or fragmentation of important regions of the cell itself.

[0204] Such recombinant bacteria or Listeria strains can be produced by transforming a bacterium or Listeria strain, or an attenuated bacterium or Listeria strain described elsewhere herein, with a plasmid or vector containing a nucleic acid encoding a recombinant fusion polypeptide. The plasmid may be an episomal plasmid that is not integrated into the host chromosome. Alternatively, the plasmid may be an integrative plasmid that integrates into the chromosome of the bacterium or Listeria strain. The 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. For example, 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; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.; and Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. are hereby incorporated by reference in their entirety for all purposes.

[0205] Bacteria or Listeria strains in which heterologous nucleic acids are integrated into the genome can be produced, for example, by using a site-specific integration vector, whereby bacteria or Listeria containing the integrated gene are produced using homologous recombination. The integration vector can be any site-specific integration vector capable of infecting the bacteria or Listeria strain. Such integration vectors can include, for example, the PSA attPP’ site, the gene encoding PSA integrase, the U153 attPP’ site, the gene encoding U153 integrase, the A118 attPP’ site, the 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 the bacterial or Listeria chromosome. Techniques for homologous recombination are well known and are 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 US6,855,320, each of these references being incorporated herein by reference in its entirety for all purposes.

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

[0208] Integration into the chromosome of bacteria or Listeria can also be achieved using phage integration sites (see, for example, Lauer et al. (2002) J Bacteriol 184(15):4177-4186, which is incorporated herein by reference in its entirety for all purposes). For example, using an integrase gene and a binding site of a bacteriophage (e.g., U153 or PSA Listeria phage), a heterologous gene can be inserted into the corresponding binding site, and the corresponding binding site 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 binding site used prior to the integration of the heterologous nucleic acid. By such methods, for example, a single-copy integrant can be obtained. To avoid the "phage curing step", a phage-based integration system based on the PSA phage can be used (see, for example, Lauer et al. (2002) J Bacteriol 184:4177-4186, which is incorporated herein by reference in its entirety for all purposes). Maintenance of the integrated gene may require, for example, continuous selection with an antibiotic. Alternatively, a phage-based chromosomal integration system that does not require selection with an antibiotic can be established. 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, for example, a host strain with auxotrophy for essential enzymes such as D-alanine racemase (e.g., Lm dal(-)dat(-)) is used.

[0209] Genetic material and / or plasmids can also be introduced into bacteria using conjugation. The method of conjugation is well known and is 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, each of which is hereby incorporated by reference in its entirety for all purposes.

[0210] In certain examples, the recombinant bacterium or Listeria strain can contain nucleic acid encoding a recombinant fusion polypeptide that is incorporated into the bacterium or Listeria genome as an open reading frame having an endogenous actA sequence (encoding the ActA protein) or an 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] The 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 metabolism-related genes can be used for selection instead of, or in addition to, antibiotic resistance genes. As an example, for selection of auxotrophic bacteria containing a plasmid encoding a metabolic enzyme or a complementary gene provided herein, the transformed auxotrophic bacteria can be grown in a medium in which selection is made for the expression of a gene encoding a metabolic enzyme (e.g., an amino acid metabolism-related gene) or a complementary gene. Alternatively, temperature-sensitive plasmids can be used to select recombinants, or any other known means can be used to select 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 "attenuated" encompasses a decrease in the ability of 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, as an example, when using intravenous inoculation of attenuated Listeria into BALB / c mice, the lethal dose (LD 50 ) at which 50% of the inoculated animals survive is the LD 50 of wild-type Listeria.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. Thus, an attenuated strain of Listeria either does not kill the animal to which it is administered or kills the animal only if the number of bacteria administered is very much greater than the number of wild-type non-attenuated bacteria required to kill the same animal. Attenuated bacteria should also be interpreted to mean that they cannot replicate in a general environment because the nutrients required for their growth are not present therein. Thus, the bacteria are limited to replication in a controlled environment where the necessary nutrients are provided. The attenuated strain is environmentally safe because it cannot replicate without control. (1) Method for attenuating bacteria and Listeria strains

[0213] Attenuation can be accomplished by any known means. For example, such attenuated strains may lack one or more endogenous virulence genes, or may lack one or more endogenous metabolism-related 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., an inactivating mutation). The term "mutation" includes any type of mutation or modification to a sequence (nucleic acid or amino acid sequence) and can include deletions, truncations, insertions, substitutions, disruptions, or translocations. For example, a mutation can include a frameshift mutation, a mutation that causes premature termination of a protein, or a mutation in a regulatory sequence that affects gene expression. Mutagenesis can be achieved using recombinant DNA technology or using traditional mutagenesis techniques using mutagenic chemicals or radiation and subsequent selection of mutants. In some embodiments, deletion mutants are used because the probability of reversion occurring concomitantly is low. The term "metabolism-related gene" refers to a gene that encodes an enzyme involved in or required for the synthesis of nutrients utilized or required by the host bacterium. For example, this enzyme can be involved in or required for the synthesis of nutrients necessary for the sustained growth of the host bacterium. The term "virulence" gene includes a gene whose presence or activity in an organism's genome contributes to the pathogenicity of that organism (e.g., enables the organism to establish a niche in the host (including adhesion to cells), evade immunity (evade the host's immune response), suppress immunity (inhibit the host's immune response), enter and exit cells, or acquire 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, for example, US2011 / 0142791, which is hereby incorporated by reference in its entirety for all purposes. LmddA is based on a Listeria strain that is attenuated due to a deletion of the endogenous virulence gene actA. Such strains can retain plasmids for antigen expression in vivo and in vitro due to complementation of the dal gene. Alternatively, LmddA may 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 having a partial deletion or inactivating mutation of the prfA gene. The PrfA protein controls the expression of a regulon that includes essential virulence genes required for Lm to colonize its vertebrate host. Thus, prfA mutations strongly impair 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 bacterial natural virulence - internalin B and actA - are deleted.

[0217] Other examples of attenuated bacteria or Listeria strains include bacteria or Listeria strains lacking one or more endogenous virulence genes. Examples of such genes include actA, prfA, plcB, plcA, inlA, inlB, inlC, inlJ, and bsh in Listeria. The attenuated Listeria strain may be a double mutant or triple mutant of any of the above strains. The attenuated Listeria strain may contain a mutation or deletion for each of the genes provided herein (e.g., including the actA, prfA, and dal / dat genes), or may contain a mutation or deletion for any 10 or fewer of said genes. For example, the attenuated Listeria strain may contain a mutation or deletion of the endogenous internalin C (inlC) 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 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. The translocation of Listeria to adjacent cells is inhibited by the deletion of the endogenous actA gene and / or the endogenous inlC gene or the endogenous inlB gene involved in the process, thereby resulting in an increase in immunogenicity and usefulness as a strain backbone along with a high level of attenuation. The attenuated Listeria strain may be a double mutant containing mutations or deletions of both plcA and plcB. In some cases, the strain may be constructed from the EGD Listeria backbone.

[0218] Bacteria or Listeria strains may be auxotrophic strains having mutations in metabolism-related genes. As an example, the strain may lack one or more endogenous amino acid metabolism-related genes. For example, the generation of an auxotrophic strain of Listeria lacking D-alanine can be achieved in a number of ways, which are well known and include, for example, 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 the probability of the accompanying reversion of the auxotrophic phenotype is low. As an example, mutants of D-alanine generated according to the protocols presented herein can be tested in a simple laboratory culture assay for the ability to grow in the absence of D-alanine. Mutants that are unable to 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 synthetase, D-glutamate synthase genes, D-alanine aminotransferase (dat) genes, D-alanine racemase (dal) genes, 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 lack two or more such genes (e.g., dat and dal). D-glutamate synthesis is partially controlled by the dal gene involved in the conversion of D-glu+pyr to alpha-ketoglutarate+D-ala and the reverse reaction.

[0220] As another example, attenuated Listeria strains may lack endogenous synthase genes such as amino acid synthesis genes. Examples of such genes include folP, genes encoding dihydrouridine synthase family proteins, ispD, ispF, genes encoding phosphoenolpyruvate synthase, hisF, hisH, fliI, genes encoding ribosomal large subunit pseudouridine synthase, ispD, genes encoding bifunctional GMP synthase / glutamine amidotransferase proteins, cobS, cobB, cbiD, genes encoding uroporphyrin-III C-methyltransferase / uroporphyrinogen-III synthase, cobQ, uppS, truB, dxs, mvaS, dapA, ispG, folC, genes encoding citrate synthase, argJ, genes encoding 3-deoxy-7-phosphoheptulonate synthase, genes encoding indole-3-glycerol phosphate synthase, genes encoding anthranilate synthase / glutamine amidotransferase components, menB, genes encoding menaquinone-specific isochorismate synthase, genes encoding phosphoribosylformylglycinamidine synthase I or II, genes encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, carB, carA, thyA, mgsA, aroB, hepB, rluB, ilvB, ilvN, alsS, fabF, fabH, genes encoding pseudouridine synthase, pyrG, truA, pabB, and ATP synthase genes (e.g., atpC, atpD-2, aptG, atpA-2, etc.).

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

[0222] Other attenuated bacteria and Listeria strains may lack an endogenous metabolic enzyme used in the bacterial growth process, replication process, cell wall synthesis, protein synthesis, fatty acid metabolism, or any other growth or replication process, or an amino acid metabolized in any other growth or replication process. Similarly, the attenuated strain may lack an endogenous metabolic enzyme capable of catalyzing the formation of an amino acid used in cell wall synthesis, an endogenous metabolic enzyme capable of catalyzing the synthesis of an amino acid used in cell wall synthesis, or an endogenous metabolic enzyme capable of participating in the synthesis of an amino acid used in cell wall synthesis. Alternatively, an amino acid may be used in cell wall biosynthesis. Alternatively, the metabolic enzyme is an enzyme for synthesizing D-glutamic acid, which is a cell wall component.

[0223] Other attenuated Listeria strains may be deficient in the D-glutamate synthetase gene, dga, a metabolic enzyme encoded by the alr (alanine racemase) gene, or any other enzyme involved in alanine synthesis. Still other 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);Enzymes encoded by genes involved in the synthesis of the cell wall component diaminopimelic acid ((S)-4-amino-5-oxopentanoate to 5-aminolevulinate formation catalyzed by), the enzyme encoded by gsaB - glutamate-1-semialdehyde aminotransferase ((S)-4-amino-5-oxopentanoate to 5-aminolevulinate formation catalyzed by), the enzyme encoded by hemL ((S)-4-amino-5-oxopentanoate to 5-aminolevulinate formation catalyzed by), the enzyme encoded by aspB (aspartate aminotransferase catalyzing the formation of oxalozcetate and L-glutamate from L-aspartate and 2-oxoglutarate), the enzyme encoded by argF-1 (involved in arginine biosynthesis), the enzyme encoded by aroE (involved in amino acid biosynthesis), the enzyme encoded by aroB (involved in 3-dehydroquinic acid biosynthesis), the enzyme encoded by aroD (involved in amino acid biosynthesis), the enzyme encoded by aroC (involved in amino acid biosynthesis), the enzyme encoded by hisB (involved in histidine biosynthesis), the enzyme encoded by hisD (involved in histidine biosynthesis), the enzyme encoded by hisG (involved in histidine biosynthesis), the enzyme encoded by metX (involved in methionine biosynthesis), the enzyme encoded by proB (involved in proline biosynthesis), the enzyme encoded by argR (involved in arginine biosynthesis), the enzyme encoded by argJ (involved in arginine biosynthesis), thil (involved in thiamine biosynthesis), the enzyme encoded by LMOf2365_1652 (involved in tryptophan biosynthesis), the enzyme encoded by aroA (involved in tryptophan biosynthesis), the enzyme encoded by ilvD (involved in valine and isoleucine biosynthesis), the enzyme encoded by ilvC (involved in valine and isoleucine biosynthesis), the enzyme encoded by leuA (involved in leucine biosynthesis), the enzyme encoded by dapF (involved in lysine biosynthesis), and the enzyme encoded by thrB (involved in threonine biosynthesis) (all with GenBank accession number NC_002973);

[0224] Attenuated Listeria strains can also be generated by mutations in other metabolic enzymes such as tRNA synthetases. For example, the metabolic enzyme can be encoded by the trpS gene that encodes tryptophanyl-tRNA synthetase. For example, the host strain bacteria can be Δ(trpS aroA), and both markers may be contained in the 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 cell wall teichoic acid glycosylation protein GtcA.

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

[0227] Still other examples of metabolic enzymes that can be mutated to generate attenuated Listeria strains include metabolic enzymes encoded by murG, murD, murA-1 or murA-2 (all described in GenBank accession number NC_002973). Alternatively, the metabolic enzyme can be any other synthase of peptidoglycan components or precursors. The metabolic enzyme can also be a trans-glycosylase, a trans-peptidase, a 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] By mutating the corresponding orthologous genes in other bacterial strains, other bacterial strains can be attenuated as described above for Listeria. (2) Methods of complementing attenuated bacteria and Listeria strains

[0229] The attenuated bacteria or Listeria strains disclosed herein can further comprise a nucleic acid comprising a complementing gene, or a nucleic acid 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 complementing gene or encoding a complementing metabolic enzyme. Alternatively, a first nucleic acid can encode a fusion polypeptide, and another second nucleic acid can comprise a complementing gene or can encode a complementing metabolic enzyme.

[0230] Complementary genes can be extrachromosomal or integrated into the bacterial or Listeria genome. For example, auxotrophic Listeria strains can contain episomal plasmids that contain nucleic acids encoding metabolic enzymes. Such plasmids will be contained in Listeria in an episomal or extrachromosomal manner. Alternatively, auxotrophic Listeria strains can contain integrative plasmids (i.e., integration vectors) that contain nucleic acids encoding metabolic enzymes. Such integrative plasmids can be used for integration into the Listeria chromosome. In some embodiments, the episomal plasmid or integrative plasmid lacks an antibiotic resistance marker.

[0231] Instead of or in addition to an antibiotic resistance gene, metabolic-related genes can be used for selection. As an example, for selection of auxotrophic bacteria containing a plasmid encoding a metabolic enzyme or complementary gene provided herein, transformed auxotrophic bacteria can be grown in a medium in which selection is for the expression of a gene encoding a metabolic enzyme (e.g., an amino acid metabolism-related gene) or complementary gene. For example, bacteria that are auxotrophic for D-glutamate synthesis can be transformed with a plasmid containing a gene for D-glutamate synthesis, and the auxotrophic bacteria will grow in the absence of D-glutamate, whereas auxotrophic bacteria not transformed with said plasmid and auxotrophic bacteria that do not express a plasmid encoding a protein for D-glutamate synthesis will not grow. Similarly, bacteria that are auxotrophic for D-alanine synthesis will grow in the absence of D-alanine if transformed and expressing a plasmid containing a nucleic acid encoding an amino acid metabolic enzyme for D-alanine synthesis. Such methods for making appropriate media containing or lacking the necessary growth factors, nutritional supplements, amino acids, vitamins, antibiotics, etc. are well known and such methods are commercially available.

[0232] If auxotrophic bacteria containing a plasmid encoding a metabolic enzyme or a complementary gene provided in this specification are selected in an appropriate medium, they can be propagated in the presence of a selection pressure. Such propagation can include the growth of the bacteria in a medium that does not contain auxotrophic factors. The presence of the plasmid expressing the metabolic enzyme or the complementary gene in the auxotrophic bacteria ensures that the plasmid will replicate with the bacteria, and thus, the bacteria carrying the plasmid will be continuously selected. The production of 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 growing.

[0233] In one particular example, the attenuated strain is a strain having 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 may have the sequence set forth in SEQ ID NO: 76 (encoded by SEQ ID NO: 78; GenBank accession number AF038438), or may 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 may also be any other Listeria alanine racemase protein. Alternatively, the alanine racemase protein may be any other gram-positive alanine racemase protein, or any other alanine racemase protein. An exemplary D-amino acid aminotransferase protein may have the sequence set forth in SEQ ID NO: 77 (encoded by SEQ ID NO: 79; GenBank accession number AF038439), or may 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 may also be any other Listeria D-amino acid aminotransferase protein. Alternatively, the D-amino acid aminotransferase protein may 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 having a deletion or inactivating mutation in prfA (e.g., Lm prfA(-)), and the complementing gene encodes the PrfA protein. For example, the complementing gene may encode a mutant PrfA (D133V) protein that restores some PrfA function. An example of the 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 the 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 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 the function of the Listeria strain.

[0236] Other auxotrophic strains and complementation systems can also be employed for use in connection 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 include a PEST-containing peptide fused to one or more disease-associated antigenic peptides. Other such recombinant fusion polypeptides can include one or more disease-associated antigenic peptides, and the fusion polypeptide does not include a PEST-containing peptide.

[0238] Another example of a recombinant fusion polypeptide includes, from the N-terminus to the C-terminus, a bacterial secretion sequence, ubiquitin (Ub) protein, and one or more disease-associated antigen peptides (i.e., in series, e.g., Ub-peptide 1-peptide 2). Alternatively, when two or more disease-associated antigen peptides are used, each antigen peptide may be fused to its own secretion sequence and Ub protein (e.g., Ub1-peptide 1; Ub2-peptide 2), and a combination of separate fusion polypeptides may be used.

[0239] Nucleic acids encoding such recombinant fusion polypeptides (referred to as mini-gene constructs) are also disclosed. Such mini-gene nucleic acid constructs can further include two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, the mini-gene nucleic acid construct can further include 2 to 4 open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different peptide. In some nucleic acid constructs, two stop codons may follow the codon encoding the carboxy terminus of the fusion polypeptide to ensure termination of protein synthesis.

[0240] The bacterial signal sequence can be a signal sequence of Listeria, such as the Hly or ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be the LLO signal sequence. An exemplary LLO signal sequence is set forth in SEQ ID NO: 97. The signal sequence can be of bacterial origin, specific to the host bacterium (e.g., Listeria monocytogenes, e.g., the secA1 signal peptide), or heterologous 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; secA2 signal peptides such as the p60 signal peptide from Listeria monocytogenes; and Tat signal peptides such as the B. subtilis Tat signal peptide (e.g., PhoD). In certain examples, the secretion signal sequence is from a Listeria protein, e.g., ActA 300 secretion signal, or ActA 100 is a secretion signal. An exemplary ActA signal sequence is set forth in SEQ ID NO: 98.

[0241] Ubiquitin can be, for example, a full-length protein. Ubiquitin expressed from the nucleic acid constructs provided herein can be cleaved by hydrolysis at the carboxy terminus from the remainder of the recombinant fusion polypeptide expressed from the nucleic acid construct upon entry into the host cell cytosol. This cleavage releases the amino terminus of the fusion polypeptide, resulting in the production of the peptide in the host cell cytosol.

[0242] The selection, variability, and composition of the antigenic peptides in the fusion polypeptide are discussed in detail elsewhere herein, and examples of disease-related antigenic peptides are discussed in more detail elsewhere herein.

[0243] The recombinant fusion polypeptide can contain one or more tags. For example, the recombinant fusion polypeptide can contain one or more peptide tags on the N-terminal and / or C-terminal side of one or more antigenic peptides. The tag can be directly fused to the antigenic peptide or linked to the antigenic peptide via a linker (examples of which are disclosed elsewhere in this specification). Examples of tags include the following: FLAG tag, 2×FLAG tag, 3×FLAG tag, His tag, 6×His 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 3×FLAG 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 3×FLAG tag is set forth in SEQ ID NO: 99. Two or more tags, for example, 2×FLAG tag and SIINFEKL tag, 3×FLAG tag and SIINFEKL tag, or 6×His tag and SIINFEKL tag, can be used together. When two or more tags are used, they can be located at any position and in any order within the recombinant fusion polypeptide. For example, two tags can be at the C-terminus of the recombinant fusion polypeptide, two tags can be at the N-terminus of the recombinant fusion polypeptide, two tags can be located internally within the recombinant fusion polypeptide, there can be one tag at the C-terminus and one tag at the N-terminus of the recombinant fusion polypeptide, there can be one tag at the C-terminus and one tag internally within the recombinant fusion polypeptide, or there can be one tag at the N-terminus and one tag internally 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). A particular recombinant fusion polypeptide contains a C-terminal SIINFEKL tag.By such tags, it may be possible to easily detect a recombinant fusion protein, confirm the secretion of the recombinant fusion protein, or track the immunogenicity of the secreted fusion polypeptide by tracking the immune response to these "tag" array peptides. Such an immune response can be monitored using a number of reagents, for example, monoclonal antibodies specific for these tags and DNA or RNA probes.

[0244] The recombinant fusion polypeptides disclosed herein may be expressed by recombinant Listeria strains or may be expressed and isolated from other vectors and cell lines used for protein expression and isolation. Recombinant Listeria strains having expression of such antigenic peptides can be used, for example, in immunogenic compositions containing such recombinant Listeria and in vaccines containing a recombinant Listeria strain and an adjuvant. Expression of one or more antigenic peptides as fusion polypeptides having a non-hemolytic truncated form of LLO, ActA or PEST-like sequences in the host cell line of the Listeria strain and in host cell lines 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. The nucleic acid may contain DNA or RNA, or may consist of DNA or RNA, and may be single-stranded or double-stranded. The nucleic acid may be in the form of a plasmid, such as an episomal plasmid, a multi-copy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid may be in the form of a viral vector, a phage vector, or may be within a bacterial artificial chromosome. Such nucleic acids may have one open reading frame, or may have 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 between each open reading frame two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence. For example, the nucleic acid can contain between each open reading frame 2 to 4 open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence. Each open reading frame can encode a different polypeptide. In some nucleic acids, following the codon encoding the carboxy terminus of the fusion polypeptide, two stop codons follow to ensure termination of protein synthesis. A. Antigenic peptide

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

[0247] The fusion polypeptide may contain a single antigen peptide or may contain two or more antigen peptides. Each antigen peptide can be of any length sufficient to induce an immune response, and the antigen peptides may be of the same length or may have different lengths. For example, the antigen peptides disclosed herein may be 5-100, 15-50, or 21-27 amino acids in length, or may be 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 15-21, 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 in length. For example, the antigen peptide 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 antigen peptides are 21 or 27 amino acids in length. Other antigen peptides can be full-length proteins or fragments thereof.

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

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

[0250] Recurrent cancer mutations can also be from cancer-related proteins. The term "cancer-related protein" includes proteins having mutations that occur in multiple types of cancer, proteins having mutations that occur in multiple subjects having a particular type of cancer, or proteins having mutations that correlate with the occurrence or progression of one or more types of cancer. For example, a cancer-related protein can be an oncogenic protein (i.e., a protein having an activity that can contribute to cancer progression, such as 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, for example, by negative regulation of the cell cycle or by promoting apoptosis). In some embodiments, the cancer-related protein has "mutation hotspots". A mutation hotspot is an amino acid position within a protein-coding gene where mutations are seen at a higher frequency than expected in the absence of selection (e.g., somatic substitution rather than somatic aberration, e.g., by translocation, amplification, and deletion). Such hotspot mutations can occur across multiple types of cancer and / or can be shared among multiple patients. Mutation hotspots exhibit a selection pressure across a population of tumor samples. The tumor genome contains recurrent cancer mutations that "drive" tumorigenesis by acting on genes (i.e., tumor driver genes) that confer a selective growth advantage to tumor cells upon alteration. Such tumor driver genes can be identified, for example, by identifying genes in which mutations are seen at a higher frequency than expected from the background mutation rate (i.e., recurrently); by identifying genes that exhibit other positive selection signals across tumor samples (e.g., a high non-silent mutation rate compared to silent mutations, or a bias towards the accumulation of functional mutations); by taking advantage of the tendency for mutations in a particular region of the protein sequence to persist based on the finding that inactivating mutations are distributed along the protein sequence but gain-of-function mutations tend to occur specifically at certain residues or domains; or by taking advantage of the overrepresentation of mutations in specific functional residues such as phosphorylation sites.Many of these mutations often occur in the functional regions of bioactive proteins (e.g., kinase domains or binding domains) or block the active sites (e.g., phosphorylation sites), resulting in loss-of-function or gain-of-function mutations, or they can occur in a way that does not disrupt the three-dimensional structure and / or charge balance of the protein enough to interfere with normal function. Genome analysis of a large number of tumors indicates that mutations often occur at a limited number of amino acid positions. Thus, the majority of common mutations can be presented by a relatively small number of potential tumor-associated antigens or T cell epitopes.

[0251] "Recurrent cancer mutations" are changes in the amino acid sequence of a protein that occur in multiple types of cancer and / or in multiple subjects with a particular type of cancer. Such mutations associated with cancer can result in the generation of tumor-associated antigens that are not normally present in the corresponding healthy tissues.

[0252] Tumor driver genes and cancer-related 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. For example, Chang et al. (2016) Nat Biotechnol 34(2):155-163; Tamborero et al. (2013) Sci Rep 3:2650 are hereby incorporated by reference in their entirety, and each of these references is incorporated herein by reference in its entirety.

[0253] As another example, the antigen peptide can be a heteroclitic antigen peptide. For example, the heteroclitic antigen peptide can be a fragment of a cancer-related protein (i.e., a continuous sequence of amino acids from a cancer-related protein) that contains a heteroclitic mutation. The heteroclitic antigen peptide may contain a single heteroclitic mutation or may 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 is derived (e.g., a peptide that does not contain an anchor residue mutation).

[0254] Some of the recombinant fusion polypeptides disclosed herein can include any combination of antigen peptides that contain recurrent cancer mutations, antigen peptides that contain heteroclitic mutations (e.g., from cancer-related proteins), and antigen peptides expressed from minigene constructs (e.g., from cancer-related proteins) (i.e., antigen peptides such as heteroclitic antigen peptides fused to ubiquitin). For example, such a recombinant fusion polypeptide can include a PEST-containing peptide fused to two or more antigen peptides, where at least one antigen peptide is from a cancer-related protein, contains a recurrent cancer mutation, and at least one antigen peptide is from a cancer-related protein and contains a heteroclitic mutation. Optionally, the PEST-containing peptide includes a bacterial secretion signal sequence, the fusion polypeptide further includes a ubiquitin protein fused to the carboxy-terminal antigen peptide, and the PEST-containing peptide, the two or more antigen peptides, the ubiquitin, and the carboxy-terminal antigen peptide are arranged in series from the amino terminus to the carboxy terminus of the fusion protein.

[0255] Each antigenic peptide may also be hydrophilic or may have a score at most a certain hydrophobicity threshold or less that predicts secretability in Listeria monocytogenes or another bacterium of interest. For example, the antigenic peptides can be scored by a Kyte and Doolittle hydrophobicity index 21 amino acid window, and all those having a score above a cutoff (approximately 1.6) can be excluded. This is because they are less likely to be secreted by Listeria monocytogenes. Similarly, a combination of antigenic peptides or fusion polypeptides may be hydrophilic or may have a score at most a certain hydrophobicity threshold or less that predicts secretability in Listeria monocytogenes or another bacterium of interest.

[0256] The antigenic peptides can be linked to each other in any manner. For example, the antigenic peptides can be directly fused to each other without an intervening sequence. Alternatively, the 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 a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides.

[0257] Any suitable array can be used for the peptide linker. As an example, the linker array can be, for example, from 1 to about 50 amino acids in length. Some linkers can be hydrophilic. The linker can serve various purposes. For example, the linker can increase bacterial secretion, facilitate antigen processing, increase the flexibility of the fusion polypeptide, increase the rigidity of the fusion polypeptide, or serve any other purpose. In some cases, to minimize repeats, different amino acid linker sequences are distributed between antigenic peptides, or different nucleic acids encoding the same amino acid linker sequence are distributed between antigenic peptides (e.g., SEQ ID NOs: 84-94). This can also help to enable sufficient transcription, translation, secretion, maintenance, or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within the Lm recombinant vector strain population by reducing secondary structure. Other suitable peptide linker sequences can be selected, for example, based on 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 functional epitopes on the antigenic peptide, and (3) the absence of hydrophobic or charged residues that might react with functional epitopes. 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; US4,935,233; and US4,751,180, each of which is hereby incorporated 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, may be used in the state of a linker containing an array repetition, or may be used in a linker further containing one or more of the other arrays in the table), but other ones are also conceivable (for example, see Reddy Chichili et al. (2013) Protein Science 22:153-167, which is incorporated herein by reference in its entirety for all purposes). Unless otherwise specified, "n" represents the undetermined number of repetitions in the listed linker.

[0258]

Table 2

[0259] The recombinant fusion proteins disclosed herein include PEST-containing peptides. The PEST-containing peptide may be at the amino terminus (N-terminus) of the fusion polypeptide (i.e., the N-terminus of the antigen peptide), may be at the carboxy terminus (C-terminus) of the fusion polypeptide (i.e., the C-terminus of the antigen peptide), or may be embedded in the antigen peptide. In some recombinant Listeria strains and methods, the PEST-containing peptide is separate from the fusion polypeptide rather than being part of the fusion polypeptide. Fusion of an antigen peptide such as an LLO peptide to a PEST-like sequence can enhance the immunogenicity of the antigen peptide and increase the cell-mediated antitumor immune response (i.e., increase the cell-mediated antitumor immunity). For example, see Singh et al. (2005) J Immunol 175(6):3663-3673, which is incorporated herein by reference in its entirety for all purposes.

[0260] PEST-containing peptides are those that contain a PEST sequence or a PEST-like sequence. The PEST sequence in eukaryotic proteins has been identified quite some time ago. For example, proteins containing an amino acid sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST), which are not always but generally adjacent to a cluster containing several positively charged amino acids, have a rapid intracellular half-life (Rogers et al. (1986) Science 234:364-369, which is hereby incorporated by reference in its entirety for all purposes). Furthermore, these sequences have been reported to target proteins to the ubiquitin-proteasome pathway for degradation (Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21:267-271, which is hereby incorporated by reference in its entirety for all purposes). This pathway is also used by eukaryotic cells to generate immunogenic peptides that bind to MHC class I, and a hypothesis has been put forward that PEST sequences are present in large amounts in eukaryotic proteins that give rise to immunogenic peptides (Realini et al. (1994) FEBS Lett. 348:109-113, which is hereby incorporated by reference in its entirety for all purposes). Prokaryotic proteins usually do not contain a PEST sequence. This is because these proteins do not have 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 has been reported to be essential for L. monocytogenes pathogenicity (Decatur and Portnoy (2000) Science 290:992-995, which is hereby incorporated by reference in its entirety for all purposes). The presence of this PEST-like sequence in LLO targets the protein for degradation by the proteolytic machinery of the host cell, so that once LLO has fulfilled its function and facilitated the escape of L. monocytogenes from the vacuole of the phagosome or phagolysosome, the protein is destroyed before it can damage the cell.

[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 in Rechsteiner and Rogers (1996) Trends Biochem.Sci.21:267-271, each of these references being hereby incorporated by reference in its entirety for all purposes. A PEST or PEST-like sequence can be identified using a PEST discovery program. For example, a PEST-like sequence can be a region rich in proline (P), glutamate (E), serine (S) and threonine (T) residues. Optionally, one or more clusters containing several positively charged amino acids can be adjacent to the PEST-like sequence. For example, a PEST-like sequence can be defined as a hydrophilic stretch of at least 12 amino acids in length having a high local concentration of proline (P), aspartate (D), glutamate (E), serine (S) and / or threonine (T) residues. In some cases, a PEST-like sequence does 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, a PEST-like sequence conforms to the algorithm disclosed in Rogers et al. In another example, a PEST-like sequence conforms to the algorithm disclosed in Rechsteiner and Rogers. A PEST-like sequence can also be identified by first scanning for the positively charged amino acids R, H and K within the specified protein sequence. All amino acids flanked by positive charges are counted and only motifs containing an equal or greater number of amino acids than the window size parameter are further considered. Optionally, a PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T.

[0263] The quality of the PEST motif can be improved by scoring parameters based on the local concentration of important amino acids and the hydrophobicity of the motif. The concentrations of D, E, P, S, and T are expressed as mass percentages (w / w) and are corrected for one equivalent of D or E, one equivalent of P, and one equivalent of S or T. The calculation of hydrophobicity may also in principle follow the method of Kyte and Doolittle (1982) J. Mol. Biol. 157:105, which is hereby incorporated by reference in its entirety for all purposes. For simplicity of calculation, the Kyte–Doolittle hydropathy index, which originally ranges from −4.5 for arginine to +4.5 for isoleucine, is converted to a positive integer using the following linear transformation, which gives values from 0 for arginine to 90 for isoleucine: hydropathy index = 10 × Kyte–Doolittle hydropathy 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 the hydrophobicity index for each amino acid species. The desired PEST score is obtained as a combination of a term for local concentration and a term for hydrophobicity, 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] It is also possible to identify PEST-like sequences using any other known available method or algorithm. For example, see CaSPredictor (Garay-Malpartida et al. (2005) Bioinformatics 21 Suppl 1:i169-76, which is hereby incorporated by reference in its entirety for all purposes). Another method that can be used is as follows: a 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 of the PEST residues is 1, and the CV for each of the other AAs (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 US7,635,479, US7,665,238, and US2014 / 0186387, each of these patent references being hereby incorporated by reference in its entirety for all purposes.

[0268] The PEST-like sequence can be from a Listeria species, for example, from 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. The streptolysin O protein from Streptococcus sp. also contains a PEST sequence. For example, Streptococcus pyogenes streptolysin O contains the PEST sequence KQNTASTETTTTNEQPK (SEQ ID NO: 49) at amino acids 35-51, and Streptococcus equisimilis streptolysin O contains the PEST-like sequence KQNTANTETTTTNEQPK (SEQ ID NO: 50) at amino acids 38-54. Another example of a PEST-like sequence is from the Listeria seeligeri cytolysin encoded by the lso gene: RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 51).

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

[0270] An 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 the LLO protein is the protein designated by GenBank accession number P13128 (SEQ ID NO: 55; the nucleic acid sequence is described in GenBank accession number 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, and when it is secreted by bacteria and cleaved from LLO, a 504-amino acid full-length active LLO protein without the signal sequence results. The LLO peptides disclosed herein may include the signal sequence or may include peptides without the signal sequence. Exemplary LLO proteins that can be used include 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 thereof, or consist essentially of such, or consist of such. Any sequence encoding a fragment of the LLO protein, or a homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant, or fragment of an analog of the LLO protein can be used. Homologous LLO proteins can have a sequence identity with a reference LLO protein of, for example, higher than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98% or 99%.

[0271] Another example of the LLO protein is set forth in SEQ ID NO: 56. The LLO proteins that can be used may include 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 thereof, or may consist essentially of such, or may consist of such.

[0272] Another example of the LLO protein is the LLO protein from the Listeria monocytogenes 10403S strain, such as that described by GenBank accession number ZP_01942330 or EBA21833, or encoded by the nucleic acid sequences described by GenBank accession number NZ_AARZ01000015 or AARZ01000015.1. Another example of the LLO protein is the LLO protein from the Listeria monocytogenes 4b F2365 strain (see, for example, GenBank accession number YP_012823), the LLO protein from the EGD-e strain (see, for example, GenBank accession number NP_463733), or the LLO protein from any other strain of Listeria monocytogenes. Yet another example of the LLO protein is the LLO protein from the Flavobacteriales bacterium HTCC2170 (see, for example, GenBank accession number ZP_01106747 or EAR01433, or encoded by GenBank accession number NZ_AAOC01000003). The LLO protein that can be used can include any of the above LLO proteins, or homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms thereof, can consist essentially of such, or can consist of such.

[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 alveolysin, which can be found, for example, in Paenibacillus alvei (see, for example, GenBank accession number P23564 or AAA22224, or encoded by GenBank accession number 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 may retain one or more functionalities of the native LLO protein, or may lack one or more functionalities of the native LLO protein. For example, the retained LLO functionality can be enabling escape from phagosomes or phagolysosomes by bacteria (e.g., Listeria), or enhancing the immunogenicity of the peptide to which it is fused. The retained functionality can also be a hemolytic function or an antigenic function. Alternatively, the LLO peptide can be non-hemolytic LLO. Other functions of LLO are known, and methods and assays for evaluating LLO functionality are also known.

[0275] The LLO fragment may be a PEST-like sequence or may contain a PEST-like sequence. The LLO fragment can include one or more of internal deletions, truncations from the C-terminus, and truncations from the N-terminus. In some cases, the LLO fragment can include more than one internal deletion. Other LLO peptides can be full-length LLO proteins having 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, the LLO protein can be made non-hemolytic by deletion or mutation of the activation domain at the carboxy terminus, by deletion or mutation of cysteine 484, or by deletion or mutation at another position.

[0277] Other LLO proteins are made non-hemolytic by deletion or mutation of the cholesterol binding domain (CBD) as detailed in US8,771,702, which is hereby incorporated by reference in its entirety for all purposes. Mutations can include, for example, substitutions or deletions. 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 a mutation 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 a mutation of residues that align optimally with SEQ ID NO: 55 (e.g., matching cysteine or tryptophan residues). By way of example, a mutant LLO protein can be created in which residues C484, W491, and W492 of LLO are substituted with alanine residues, and this substitution will result in a substantial decrease in hemolytic activity compared to wild-type LLO. A mutant LLO protein having the C484A, W491A, and W492A mutations is designated "mutLLO".

[0278] As another example, mutant LLO proteins having internal deletions that include cholesterol-binding domains can be generated. The sequence of the cholesterol-binding domain of SEQ ID NO: 55, set forth in SEQ ID NO: 74. For example, the internal deletion can be a 1-11 amino acid deletion, an 11-50 amino acid deletion, or longer. Similarly, the mutated region can 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 will result in a deleted sequence that includes the CBD (residues 483-493 of SEQ ID NO: 55). However, the mutated region can be a fragment of the CBD or can 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, which will result in a substantial decrease in hemolytic activity compared to wild-type LLO.For example, CBD (ECTGLAWEWWR; SEQ ID NO: 74) can be replaced with the CTL epitope (ESLLMWITQCR; SEQ ID NO: 75) from the antigen NY-ESO-1, which contains the HLA-A2 restricted epitope 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 be replaced by an equal number of heterologous amino acids, a smaller number of heterologous amino acids, or a larger 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, 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 1 residue, 2 residues, 3 residues, or more residues). The mutated residues may or may not be contiguous.

[0280] In one exemplary 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 are 492 - 528 amino acids in length. For example, the LLO fragment can consist of the first approximately 440 or 441 amino acids of the LLO protein (e.g., the first 441 amino acids of SEQ ID NO: 55 or 56, or a fragment that aligns optimally 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 the LLO protein (e.g., the first 420 amino acids of SEQ ID NO: 55 or 56, or a fragment that aligns optimally with SEQ ID NO: 55 or 56 of another LLO protein). Other N-terminal fragments can consist of approximately 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 aligns optimally with SEQ ID NO: 55 or 56 of another LLO protein). Other N-terminal LLO fragments include any ΔLLO without an activation domain containing cysteine 484, in particular, without cysteine 484. For example, the 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 the 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 aligns optimally with SEQ ID NO: 55 or 56 of another LLO protein). In some embodiments, the fragment contains one or more PEST-like sequences. The LLO fragment and the truncated LLO protein can contain residues of a homologous LLO protein corresponding to any one of the specific amino acid ranges described above. The number of residues need not exactly match the number of residues listed above (e.g., if the homologous LLO protein has insertions or deletions compared to the specific LLO proteins disclosed herein).Examples of N-terminal LLO fragments include SEQ ID NOs: 57, 58, and 59. The LLO proteins that can be used include the sequences 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 thereof, or consist essentially of such, or consist of such. In some compositions and methods, the N-terminal LLO fragment set forth in SEQ ID NO: 59 is used. 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-bound protein that acts as a scaffold to promote the polymerization, assembly, and activation of host actin polymers to propel Listeria monocytogenes through the cytoplasm in infected host cells. Immediately after entry into the mammalian cell cytosol, L. monocytogenes induces the polymerization of host actin filaments and uses the forces generated by actin polymerization to first move intracellularly and then from cell to cell. ActA is involved in actin nucleation and the mediation of actin-based motility. The ActA protein acts as a scaffold for assembling the cell's actin polymerization machinery by providing multiple binding sites for host cell cytoskeletal components. The N-terminus of ActA binds monomeric actin and acts as a constitutively active nucleation-promoting factor by stimulating the endogenous actin nucleation activity. Both the actA and hly genes are members of a 10 kb gene cluster regulated by the transcriptional activator PrfA, and actA is approximately 226-fold upregulated in the mammalian cytosol. Any sequence encoding the ActA protein, or a homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant or fragment of an analog of the ActA protein can be used. The homologous ActA protein can have a sequence identity with the reference ActA protein that is higher than, for example, 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98% or 99%.

[0283] An example of the ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 61. Another example of the 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 a signal sequence that is cleaved from the ActA protein when the proprotein is secreted by the bacterium. The ActA peptide may or may not contain a signal sequence (e.g., amino acids 1-29 of SEQ ID NO: 61 or 62). Other examples of the ActA protein include, consist essentially of, or consist of homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of isoforms, or fragments of analogs of SEQ ID NO: 61 or 62.

[0284] Another example of the ActA protein is 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 NCTC strain 5348 (GenBank accession number EU394961), the ActA protein from NICPBP strain 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 protein that can be used can include, consist essentially of, or consist of any of the above LLO proteins, or homologs, variants, isoforms, analogs, fragments, fragments of homologs, fragments of variants, fragments of analogs, and fragments of isoforms thereof.

[0285] The ActA peptide can be a full-length ActA protein, or 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 comprises at least one PEST sequence (e.g., more than one PEST sequence). Additionally, the truncated ActA protein can optionally comprise an ActA signal peptide. Examples of PEST-like sequences contained in the truncated ActA protein include SEQ ID NOs: 45-48. Some such truncated ActA proteins comprise at least two or their homologs of the PEST-like sequences set forth in SEQ ID NOs: 45-48, at least three or their homologs of the PEST-like sequences set forth in SEQ ID NOs: 45-48, or all four or their homologs of the PEST-like sequences set forth in SEQ ID NOs: 45-48. Examples of truncated ActA proteins include those comprising about residues 30-122, about residues 30-229, about residues 30-332, about residues 30-200, or about residues 30-399 of the full-length ActA protein sequence (e.g., SEQ ID NO: 62), consisting essentially of such residues, or consisting of such residues. Other examples of truncated ActA proteins include those comprising the first about 50, 100, 150, 200, 233, 250, 300, 390, 400 or 418 residues of the full-length ActA protein sequence (e.g., SEQ ID NO: 62), consisting essentially of such residues, or consisting of such residues. Other examples of truncated ActA proteins include those comprising about residues 200-300 or residues 300-400 of the full-length ActA protein sequence (e.g., SEQ ID NO: 62), consisting essentially of such residues, or consisting of such residues. For example, truncated ActA consists of the first 390 amino acids of the wild-type ActA protein as described in US7,655,238, which is hereby incorporated 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, which is hereby incorporated by reference in its entirety for all purposes, wherein the modified version lacks the PEST motif and contains a non-conservative QDNKR (SEQ ID NO: 73) substitution. Alternatively, the truncated ActA protein can contain residues of a homologous ActA protein corresponding to one of the amino acid ranges described above or the amino acid ranges of the ActA peptides disclosed herein. The number of residues need not exactly match the number of residues recited herein (e.g., if the homologous ActA protein has insertions or deletions compared to the ActA protein used herein, the number of residues can be adjusted accordingly).

[0286] Examples of truncated ActA proteins include, for example, the sequences set forth in SEQ ID NOs: 63, 64, 65 or 66, or proteins comprising homologs, variants, isoforms, analogs, fragments of variants, fragments of isoforms or fragments of analogs thereof, proteins consisting essentially of such, or proteins consisting of such. SEQ ID NO: 63, called ActA / PEST1, consists of amino acids 30-122 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 64, called ActA / PEST2 or LA229, consists of amino acids 30-229 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 65, called ActA / PEST3, consists of amino acids 30-332 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 66, called ActA / PEST4, 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, for example, the sequences set forth in SEQ ID NO: 67, 69, 70 or 72, or proteins comprising homologs, variants, isoforms, analogs, fragments of variants, fragments of isoforms or fragments of analogs thereof, proteins consisting essentially of such, or proteins consisting of such. 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 the Listeria monocytogenes 10403S strain. 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 Immunotherapy Constructs Encoding Recombinant Fusion Polypeptides

[0288] Also provided herein are methods of generating an immunotherapy construct encoding a recombinant fusion polypeptide disclosed herein or a composition comprising said recombinant fusion polypeptide. For example, such methods can include selecting and designing (and, for example, testing the hydrophobicity of each antigen peptide and modifying or excluding from selection an antigen peptide if it has a score above the selected hydrophobicity index threshold) antigen peptides for inclusion in the immunotherapy construct, designing one or more fusion polypeptides each comprising the selected antigen peptides, and generating a nucleic acid construct encoding the fusion polypeptide.

[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 most a certain hydrophobicity threshold or less that can predict their secretability in a particular bacterium of interest (e.g., Listeria monocytogenes). For example, antigenic peptides can be scored with a 21 - amino - acid window by the Kyte and Doolittle hydrophobicity index, and all those with a score above a cut - off (approximately 1.6) are excluded. This is because they are less likely to be secreted by Listeria monocytogenes. See, for example, Kyte - Doolittle (1982) J Mol Biol 157(1):105 - 132, which is hereby incorporated by reference in its entirety for all purposes. Alternatively, antigenic peptides with a score around the selected cut - off 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 amino acids or more. 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 cut - offs that can be used include, for example, the following ranges: 1.2 - 1.4, 1.4 - 1.6, 1.6 - 1.8, 1.8 - 2.0, 2.0 - 2.2, 2.2 - 2.5, 2.5 - 3.0, 3.0 - 3.5, 3.5 - 4.0, or 4.0 - 4.5, or the cut - off 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 can vary, for example, depending on the genus or species of the bacterium used to deliver the fusion polypeptide.

[0290] Other suitable hydrophobicity plots or other appropriate metrics are, for example, Rose et 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 those reported in Cowan and Whittaker (1990) Peptide Research 3:75-80, each of the foregoing references being hereby incorporated by reference in its entirety for all purposes.

[0291] Optionally, antigenic peptides can be scored for their ability to bind to the target human leukocyte antigen (HLA) type (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 of information include TEpredict (tepredict.sourceforge.net / help.html) or other available MHC binding metrics. For different expression vectors, e.g., Salmonella, the cut-off may vary.

[0292] Optionally, antigenic peptides can be screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing helper T epitopes, etc.) to remove the antigenic peptides from the selection or to avoid the effects of immunosuppression.

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

[0294] The selected antigenic peptides can be arrayed in one or more candidate orders of the possible fusion polypeptides. If the antigenic peptides are not only compatible with a single plasmid but also otherwise usable, priorities can be assigned to different antigenic peptides as necessary / desired, and / or different antigenic peptides can be distributed to different fusion polypeptides (e.g., for incorporation into different recombinant Listeria strains). The priorities can be determined by factors such as the relative size of the translated polypeptide, the priority of transcription, and / or the overall hydrophobicity. The antigenic peptides can be arrayed such that they are directly linked between any number of antigenic peptide pairs without any linkers or combinations of linkers, as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to include can be determined based on considerations regarding the number of constructs required for the amount of mutation, the translation and secretion efficiency of multiple epitopes from a single plasmid, and the MOI required for each bacterium or Lm containing the plasmid.

[0295] Combinations of antigenic peptides, or full fusion polypeptides (i.e., containing an antigenic peptide, a PEST-containing peptide, and any tags), that are also scored for hydrophobicity. For example, the entire fused antigenic peptide, or the full fusion polypeptide, can be scored for hydrophobicity using a sliding 21-amino acid window by the Kyte and Doolittle hydropathy index. If any region has a score above a cutoff (e.g., approximately 1.6), the antigenic peptides can be rearranged or shuffled within the fusion polypeptide until an acceptable order of the antigenic peptides (i.e., one in which no region has a score above the cutoff) is found. Alternatively, any problematic antigenic peptide can be removed, or redesigned to be of a different size. Alternatively or in addition, one or more linkers between antigenic peptides, as disclosed elsewhere herein, can be added or modified to vary the hydrophobicity. As with the hydropathy tests 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). In addition, other suitable hydropathy plots or other appropriate metrics could be used.

[0296] Optionally, combinations of antigenic peptides or full fusion polypeptides can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing helper T epitopes, etc.) to remove antigenic peptides from selection or to avoid immunosuppressive effects.

[0297] Next, nucleic acids encoding antigen peptide combination candidates or fusion polypeptides can be designed and optimized. For example, the sequence can be optimized for increased translation level, expression period, secretion level, transcription level, and any combination thereof. For example, the increase 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, a fusion polypeptide, or a nucleic acid encoding a fusion polypeptide, can be optimized for a reduction in the level of secondary structure that may be formed in the oligonucleotide sequence, or alternatively, optimized to prevent binding of any enzyme that may modify the sequence. Expression in bacterial cells may be hampered, for example, by transcriptional silencing, short mRNA half-life, secondary structure formation, binding sites for oligonucleotide-binding molecules such as repressors and inhibitors, and the availability of the rare tRNA pool. The cause of many problems related to bacterial expression lies within the original sequence. RNA optimization can include modification of cis-acting elements, adaptation of its GC content, correction of codon bias for the non-limiting tRNA pool of bacterial cells, and avoidance of internal homologous regions. Thus, optimizing the sequence may necessarily involve, for example, adjusting regions of very high (>80%) or very low (<30%) GC content. Optimizing the sequence may also necessarily involve, for example, avoiding one or more of the following cis-acting motifs: internal TATA box, chi site, and ribosome entry site; AT-rich or GC-rich sequence stretches; repetitive sequences and RNA secondary structures; (hidden) splice donor and acceptor sites; branch points; or combinations thereof. Optimizing expression may also necessarily involve adding sequence elements to adjacent regions of the gene within the plasmid and / or elsewhere.

[0299] Optimizing the array may, for example, necessarily involve adapting the codon usage frequency to the codon bias of a host gene (e.g., a Listeria monocytogenes gene). For example, codons that can 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] A nucleic acid encoding a fusion polypeptide can be generated and introduced into a delivery vehicle, e.g., a bacterial strain or a Listeria strain. Other delivery vehicles, such as vaccinia virus or virus-like particles, may be suitable for DNA immunotherapy or peptide immunotherapy. Generating a plasmid encoding the fusion polypeptide and introducing it into a bacterial strain or a Listeria strain allows culturing and characterizing the bacteria or Listeria strain 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 a lyophilized recombinant bacterium or Listeria strain as disclosed herein, wherein, optionally, the lyophilized recombinant bacterium or Listeria strain is reconstituted by dissolving it in an amount of solvent. An immunogenic composition comprising a Listeria strain may be inherently immunogenic because it contains the Listeria strain and / or the composition may further contain an adjuvant. Other immunogenic compositions include DNA immunotherapy or peptide immunotherapy compositions.

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

[0303] An immunogenic composition may comprise a single lyophilized or reconstituted recombinant bacterium or Listeria strain as disclosed herein, or may comprise a plurality of different lyophilized or reconstituted recombinant bacteria or Listeria strains as disclosed herein. A bacterium or Listeria strain comprising a first recombinant fusion polypeptide, for example, is different from a bacterium or Listeria strain comprising a second recombinant fusion polypeptide if the first recombinant fusion polypeptide comprises one antigenic peptide not contained in the second recombinant fusion polypeptide. Two recombinant fusion polypeptides may be considered different even if they contain some of the same antigenic peptides. Such different lyophilized or reconstituted recombinant bacteria or Listeria strains can be administered to a subject simultaneously or sequentially. Sequential administration is particularly useful when the drug substance, including the lyophilized or reconstituted recombinant Listeria strain (or recombinant fusion polypeptide or nucleic acid) as disclosed herein, is in a different dosage form and / or is administered on a different dosing schedule (e.g., one composition from a mixture is administered at least once a day and another is administered less frequently, e.g., once a week, once every two weeks, or once every three weeks). The plurality of lyophilized or reconstituted recombinant bacteria or Listeria strains may each contain a different set of antigenic peptides. Alternatively, two or more lyophilized or reconstituted recombinant bacteria or Listeria strains may contain 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 an antigen-presenting cell (APC), 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 may be a non-specific stimulator of the immune response or a substance that enables the formation of a depot in a subject, which, when combined with the immunogenic compositions disclosed herein, results in a more enhanced and / or long-lasting immune response. An adjuvant can advantageously act on, for example, an immune response predominantly mediated by Th1, a Th1-type immune response, or a Th1-mediated immune response. Similarly, an adjuvant can advantageously act on a cell-mediated immune response over an antibody-mediated response. Alternatively, an adjuvant can advantageously act on an antibody-mediated response. Some adjuvants can enhance the immune response by slowly releasing the antigen, while other adjuvants can mediate their effects by any of the following mechanisms: increasing cell infiltration, inflammation, and transport to the site of injection, particularly for antigen-presenting cells (APCs); promoting the activated state of APCs by upregulating co-stimulatory 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 it, IL-6 or nucleic acids encoding it, and lipopolysaccharides. Another example of a suitable adjuvant is Montanide ISA 51. Montanide ISA 51 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 can be, for example, a human protein grown in a yeast (S. cerevisiae) vector. 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 important in the binding of LLO to cholesterol and in final membrane pore formation. The three target 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 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 that is 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 the same, cell populations, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG (Bacillus Calmette-Guerin), alum, interleukins or nucleic acids encoding the same, Quil A glycoside, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins, or any other type of known adjuvant (see, e.g., Fundamental Immunology, 5th ed. (August 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, see GJV Nossal).

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

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

[0311] "Pharmaceutically acceptable carrier" refers to a vehicle that can contain an immunogenic composition and be introduced into a subject without significant adverse effects, and that does not cause adverse effects to the immunogenic composition. That is, "pharmaceutically acceptable" refers to any formulation that is safe and provides delivery suitable for the desired route of administration of at least one immunogenic composition in an effective amount for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection can be found in various readily available sources, such as Remington’s Pharmaceutical Sciences, 18th ed., 1990, which is hereby incorporated by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral) or topical application). Such pharmaceutical compositions can be buffered, for example, in which case the pH is maintained at a specific desired value in the range of 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 solutions such as saline, glucose, buffer solutions such as phosphate buffer solutions or bicarbonate buffer 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, essential oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. Pharmaceutical compositions or vaccines can also include, for example, adjuvants such as diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffers, thickening additives, lubricants, salts for affecting osmotic pressure, buffers, vitamins, coloring, flavoring, fragrances, and the like that do not react detrimentally with the immunogenic composition.

[0313] For liquid formulations (e.g., in embodiments where lyophilized recombinant bacteria or Listeria strains are reconstituted by dissolving in a certain amount of solvent), for example, pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. 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 physiological saline and buffer 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, rubber, starch (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose or dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0314] Optionally, sustained-release or controlled-release pharmaceutical compositions or vaccines can be formulated. This formulation can be achieved, for example, by the use of liposomes or compositions in which the active compound is protected by a stepwise degradable coating (e.g., by microencapsulation, multiple coatings, etc.). Such compositions can be formulated for immediate release or for sustained release. It is also possible to lyophilize the composition and use the resulting lyophilizate (e.g., in the preparation of injectable products).

[0315] The immunogenic composition, pharmaceutical composition or vaccine disclosed herein can include one or more additional compounds effective for 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, gliadel implant, hydroxycarbamide, idarubicin, 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 biologic agents, 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 stimulants, such as antibodies or functional fragments thereof that bind to T cell receptor co-stimulatory molecules, antigen-presenting cell receptors that bind to co-stimulatory molecules, or members of the TNF receptor superfamily. T cell receptor co-stimulatory molecules may include, for example, CD28 or ICOS. Antigen-presenting cell receptors that bind to co-stimulatory molecules may include, for example, CD80 receptor, CD86 receptor, or CD46 receptor. 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 Methods

[0317] The lyophilized bacteria or Listeria strains disclosed in this specification (optionally, the lyophilized recombinant bacteria or Listeria strains are restored by dissolving in a certain amount of solvent), immunogenic compositions, pharmaceutical compositions and vaccines can be used in various ways. For example, they can be used in a method of inducing or enhancing an anti-disease-related antigen (e.g., cancer-related antigen or tumor-related antigen) immune response in a subject, in a method of inducing or enhancing an anti-disease (e.g., anti-tumor or anti-cancer) immune response in a subject, in a method of treating a disease (e.g., tumor or cancer) in a subject, in a method of preventing a disease (e.g., tumor or cancer) in a subject, or in a method of protecting a subject from a disease (e.g., tumor or cancer). They can also be used in a method of increasing the ratio of effector T cells to regulatory T cells (Tregs) in the spleen and tumors of a subject, wherein the effector T cells are targeted to a disease-related antigen. They can also be used in a method of increasing disease-related antigen-specific T cells in a subject, a method of prolonging the survival period of a subject having a disease, a method of delaying the onset of a disease in a subject, or a method of alleviating the symptoms of a disease in a subject.

[0318] Methods for inducing or enhancing an anti-disease-related 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 as disclosed herein. Thereby, an anti-disease-related antigen immune response can be induced or enhanced 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 methods can also increase the ratio of effector T cells to regulatory T cells (Tregs) in the spleen and tumor microenvironment of the subject, resulting in a more pronounced anti-tumor response in the subject.

[0319] Methods 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 as disclosed herein. Thereby, an anti-disease immune response can be induced or enhanced 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-related response in the subject.

[0320] Methods for 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 as disclosed herein. As a result, the subject can initiate an immune response against the disease expressing the disease-related antigen, thereby treating the disease in the subject.

[0321] A method of preventing a disease (e.g., cancer or tumor) in a subject, or a method of protecting a subject from developing a disease can include, for example, administering to the subject a lyophilized or reconstituted recombinant bacterium or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine as disclosed herein. As a result, the subject can initiate an immune response against a disease-related antigen, thereby preventing the disease or protecting the subject from developing the disease.

[0322] In some aspects of the above methods, two or more lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered. The multiple recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered sequentially in any order or combination, or can be administered simultaneously in any combination. By way of example, if four different Listeria strains are to be administered, they can be administered sequentially, they can be administered simultaneously, or they can be administered in any combination (e.g., the first and second strains are administered simultaneously, followed by the third and fourth strains administered 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 0-10 or 3-7 days of each other. The multiple recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines may each contain a different set of antigenic peptides. Alternatively, two or more may contain the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[0323] In one embodiment, the disease is cancer or a tumor. Cancer is a physiological condition in mammals typically characterized by unregulated cell growth and proliferation. Cancer can be a hematopoietic malignancy or a solid tumor (i.e., a mass of cells resulting from excessive cell growth and proliferation, including pre-cancerous lesions). Metastatic cancer refers to cancer that has spread from the location where it first originated to another location in the body. The tumor formed by metastatic cancer cells is called a metastatic tumor or metastasis, and metastasis is also a term used to refer to the process by which cancer cells spread to other parts of the body. Generally, metastatic cancer has the same name and the same type of cancer cells as the original, or primary, cancer. Examples of solid tumors include melanoma, carcinoma, granuloma, and sarcoma. Hematological malignancies include, for example, leukemia or lymphoid malignancies such as lymphoma. Exemplary categories of cancer include the brain cancer, breast cancer, gastrointestinal cancer, genitourinary cancer, gynecological cancer, head and neck cancer, hem cancer, skin cancer, and thoracic cancer categories. Brain malignancies include, for example, glioblastoma, high-grade glioma, low-grade glioma, medulloblastoma, neuroblastoma, and pilocytic astrocytoma. Gastrointestinal cancers include, for example, colorectal cancer, gallbladder cancer, hepatocellular cancer, pancreatic cancer, PNET, gastric cancer, and esophageal cancer. Genitourinary cancers include, for example, adrenocortical cancer, bladder cancer, chromophobic renal cancer, renal (clear cell) cancer, renal (papillary) cancer, oncocytoma, 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, hypopharyngeal cancer, head and neck cancer, and adenoid cystic cancer. Hem cancers include, for example, multiple myeloma, myelodysplasia, mantle cell lymphoma, acute lymphoblastic leukemia (ALL), non-Hodgkin 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 detailed examples of such cancers include squamous cell carcinoma or cancer (e.g., oral squamous cell carcinoma), myeloma, oral cancer, juvenile nasopharyngeal angiofibroma, neuroendocrine tumor, 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 cancer 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 a brain cancer or another type of CNS or intracranial tumor. For example, the subject may have astrocytic tumors (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma), oligodendroglial tumors (e.g., oligodendroglioma, anaplastic oligodendroglioma), ependymal tumors (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixed gliomas (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma), neuroepithelial tumors of unknown origin (e.g., polar spongioblastoma, astroblastoma, cerebral gliosis), choroid plexus tumors (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 ganglioglioma, central neurocytoma, embryonal dysplastic neuroepithelial tumor, olfactory neuroblastoma), pineal parenchymal tumors (e.g., pineocytoma, pineoblastoma, pineocytoma / pineoblastoma mixed type), or tumors having mixed neuronal or neuroblastic elements (e.g., medulloepithelioma, glioblastoma, neuroblastoma, retinoblastoma, ependymoblastoma).

[0325] The term "treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, where the goal is to prevent or alleviate the symptoms of a target disease. Treating can include directly affecting the disease, directly curing, 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 metastasis of, or alleviating / improving symptoms associated with the disease, one or more of these, or combinations thereof. For example, treating can include extending the expected survival period. The effects (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 / improving symptoms of, etc.) can be effects compared to untreated or placebo-treated control subjects. The term "treating" or "treatment" can also refer to increasing the percentage of likelihood of survival or extending the expected survival period for a subject having the disease (e.g., compared to untreated or placebo-treated control subjects). In one example, "treatment" can refer to delaying progression, facilitating remission, inducing remission, increasing remission, accelerating recovery, increasing the effectiveness of alternative therapies, reducing resistance to alternative therapies, or combinations thereof. The term "preventing" or "precluding" can refer to, for example, delaying the onset of symptoms, preventing recurrence of the disease, reducing the number or frequency of recurrence episodes, extending the latency period between symptomatic episodes, or combinations thereof.The terms "suppressing" or "inhibiting" can refer to, for example, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, shortening the latency period of symptoms, improving symptoms, alleviating secondary symptoms, reducing secondary infections, prolonging patient survival, or combinations thereof.

[0326] The term "subject" refers to a mammal (e.g., a human) in need of treatment for a disease or prone to developing a disease. The term subject also refers to a mammal (e.g., a human) undergoing either a prophylactic treatment or a therapeutic treatment. The subject can include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude an individual who is healthy in all respects and has no disease or does not exhibit signs of a disease.

[0327] An individual has a high risk of developing a disease if the individual has at least one risk factor (e.g., genetic, biochemical, family history, and environmental exposure) known to statistically significantly increase the risk of developing the disease in an individual having the risk factor as compared to an individual not having the risk factor.

[0328] "Symptom" or "sign" refers to objective evidence of a disease observed by a physician or subjective evidence of a disease perceived by the subject, such as a change in gait. A symptom or sign can be any manifestation of a disease. A symptom can be either primary or secondary. The term "primary" refers to a symptom that is a direct result of a particular disease or disorder (e.g., a tumor or cancer), while the term "secondary" refers to a symptom that is derived from or results from the 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] Freeze-dried or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines are administered in an effective regimen, which means a dosage, route of administration and frequency of administration that delays the onset of at least one sign or symptom of a disease, reduces the severity, inhibits further deterioration and / or improves it. Alternatively, freeze-dried or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines are administered in an effective regimen, which means a dosage, route of administration and frequency of administration that induces an immune response against disease-related antigens in the freeze-dried or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines, or induces an immune response against the bacteria or Listeria strains themselves. When the subject is already suffering from a disease, the regimen can be called a therapeutically effective regimen. When the subject has a high risk of developing a disease compared to the general population but has not yet experienced symptoms, the regimen can be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic effectiveness can be observed in an individual patient compared to a historical control or compared to past experience in the same patient. In other cases, therapeutic or prophylactic effectiveness can be demonstrated in a population of patients treated in preclinical or clinical trials compared to a control population of untreated patients. For example, if an individual patient treated by the methods described herein achieves a better outcome than the average outcome in a control population of untreated comparator patients, or if a better outcome is demonstrated at the p<0.05 or 0.01 or even 0.001 level against control patients in a controlled clinical trial (e.g., a Phase II, Phase II / III or Phase III trial) in treated patients, the regimen can be considered therapeutically or prophylactically effective.

[0330] Exemplary dosages for recombinant Listeria strains are, for example, 1×10 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, 150~500×10 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 CFU. Other exemplary dosages for the recombinant Listeria strain are, for example, 1×10 7 organisms, 1.5×10 7 organisms, 2×10 8 organisms, 3×10 7 organisms, 4×10 7 organisms, 5×10 7 organisms, 6×10 7 organisms, 7×10 7 organisms, 8×10 7 organisms, 10×10 7 organisms, 1.5×10 8 organisms, 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 5×10 10 It is a biological agent. The dosage may depend on the patient's condition and, if any, on the response to previous treatment, whether the treatment was prophylactic or therapeutic, and on other factors.

[0331] Administration is obtained by any suitable means. For example, administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraventricular, intraperitoneal, topical, intranasal, intramuscular, intraocular, rectal, conjunctival, transdermal, intradermal, intravaginal, rectal, intratumoral, parcanceral, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal aspiration (spray), sublingual, aerosol, suppository, or a combination thereof. For intranasal administration or application by inhalation, a solution or suspension of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacterium or Listeria strain, an immunogenic composition, a pharmaceutical composition or a vaccine, which is mixed in the presence of a suitable carrier and aerosolized or nebulized, is preferred. Such an aerosol can contain any of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines described herein. Administration can also be in the form of a suppository (e.g., rectal suppository or urethral suppository), a pellet for subcutaneous implantation (e.g., providing controlled release over a period of time), or in the form of a capsule. Administration can also be by injection into the site of the disease. The dosing regimen can be readily determined based on factors such as the exact nature and type of the disease to be treated, the severity of the disease, the age and general health of the subject, the weight of the subject, the response of the individual subject, and the like.

[0332] The dosing frequency can depend, among other factors, particularly on 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 can be, for example, once a day, once a week, once a month, four times a year, or at irregular intervals, depending on changes in the condition of the subject or the progression of a tumor or cancer under treatment. The treatment course can depend on the condition of the subject and other factors. For example, the treatment course can be for several weeks, several months, or several years (e.g., up to 2 years). For example, in order to achieve regression or suppression of the disease, repeated dosing (doses) can be initiated immediately after the first treatment course or after several days, weeks or months. The assessment can 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 can be administered once every three weeks for up to 2 years. In one example, the lyophilized or reconstituted recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition or vaccine disclosed herein is administered in escalating doses to increase the effector T cell to regulatory T cell ratio and generate a more potent anti-disease immune response. For example, the anti-disease response can be further enhanced by providing cytokines to the subject, including IFN-γ, TNF-α, and other cytokines known to enhance the cellular immune response. See, for example, US 6,991,785, which is hereby incorporated by reference in its entirety 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 the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines multiple times. "Boosting" refers to administering an additional dose to the subject. For example, in some methods, 2 booster immunizations (or a total of 3 inoculations) are administered, 3 booster immunizations are administered, 4 booster immunizations are administered, 5 booster immunizations are administered, or 6 or more booster immunizations are administered. The number of dosages administered can depend, for example, on the response of the disease to the treatment.

[0334] Optionally, the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines used for booster immunizations are the same as the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines used in the first "prime" inoculation. Alternatively, the booster agent is different from the recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines of the prime immunization. Optionally, the same dosage is used for both the prime inoculation and the booster immunization. Alternatively, a greater dosage is used for the booster agent, or a lesser dosage is used for the booster agent. The period between the prime inoculation and the booster immunization can be determined based on experimentation. For example, the period between the prime inoculation and the booster immunization 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 shown to enhance immune responses and protection against a wide variety of pathogens. See, for example, 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 hereby incorporated by reference in its entirety for all purposes. By providing different forms of antigen in the prime injection and the boost injection, the immune response to the antigen can be maximized. Prime with a DNA vaccine followed by boost with protein in an adjuvant or viral vector delivery of DNA encoding the antigen is one effective way to improve antigen-specific antibody and CD4 + T cell responses or CD8 + 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 are hereby incorporated by reference in their entirety for all purposes. As an example, when a subject is vaccinated by DNA prime antigen stimulation followed by boost immunization with an adenovirus vector expressing the antigen, addition of CRL1005 poloxamer (12 kDa, 5% POE) to the DNA encoding the antigen can enhance the T cell response. See, for example, Shiver et al. (2002) Nature 415:331-335, which is hereby incorporated by reference in its entirety for all purposes. As another example, a vector construct encoding an immunogenic portion of an antigen, and a protein comprising the immunogenic portion of the antigen, can be administered. See, for example, US2002 / 0165172, which is hereby incorporated by reference in its entirety for all purposes. Similarly, the immune response to nucleic acid vaccination can be enhanced by co-administration of the polynucleotide and polypeptide of interest (e.g., within 0 to 10 or 3 to 7 days of each other in some embodiments during the same immune response). See, for example, US6,500,432, which is hereby incorporated by reference in its entirety for all purposes.

[0336] The treatment methods disclosed herein can also include the step of administering one or more additional compounds effective for the prevention or treatment of a disease (e.g., a tumor or cancer). For example, the additional compound can be a compound 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, 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 a combination thereof. Alternatively, the additional compound can also include other biologic agents, such as Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or an antibody 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 treatment 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 the administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein, may follow the administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein, or may be concurrent with the administration of the lyophilized or reconstituted recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein.

[0338] Targeted immunomodulatory therapies primarily focus on the activation of costimulatory receptors, for example, by using agonist antibodies that target members of the tumor necrosis factor receptor superfamily, including 4-1BB, OX40, and GITR (glucocorticoid-induced TNF receptor-related). Modulation of GITR has demonstrated potential in both anti-tumor and vaccine settings. Another target for agonist antibodies is costimulatory signaling molecules for T cell activation. Targeting costimulatory signaling molecules can lead to enhanced activation of T cells and promotion of a stronger immune response. Costimulation can also help prevent the inhibitory effects of checkpoint inhibition and increase antigen-specific T cell proliferation.

[0339] Listeria-based immunotherapy acts by inducing the de novo generation of tumor antigen-specific T cells that infiltrate and destroy tumors, and 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-stimulatory molecules CD137, OX40, GITR and CD40) can have a synergistic effect with Listeria-based immunotherapy.

[0340] Accordingly, some methods can further include 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 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. For example, an 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 a 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 can further include administering a T cell stimulatory substance, such as an antibody or a functional fragment thereof that binds to a T cell receptor co-stimulatory molecule, an antigen presenting cell receptor that binds to a co-stimulatory molecule, or a member of the TNF receptor superfamily. The T cell receptor co-stimulatory molecule can include, for example, CD28 or ICOS. The antigen presenting cell receptor that binds to a co-stimulatory molecule can include, for example, the CD80 receptor, the CD86 receptor, or the CD46 receptor. The TNF receptor superfamily member can include, for example, the glucocorticoid-induced 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 a functional fragment thereof that binds to a T cell receptor co-stimulatory molecule or an antibody or a functional fragment thereof that binds to an antigen presenting cell receptor that binds to a co-stimulatory molecule. The antibody can be, for example, an anti-TNF receptor antibody or an antigen-binding fragment thereof (e.g., a member of the TNF receptor superfamily such as the glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25), an anti-OX40 antibody or an antigen-binding fragment thereof, or an anti-GITR antibody or an 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 its ligand or fragment, CD137 or its ligand or fragment, or an inducible T cell costimulatory (ICOS) or its ligand or fragment, or an agonistic small molecule) can be administered.

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

[0344] Methods for assessing the effectiveness of cancer immunotherapy are well known, for example, 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 these references is hereby incorporated by reference in its entirety for all purposes. As an example, for prostate cancer, prostate cancer models such as the TRAMP-C2 mouse model, 178-2 BMA cell model, PAIII adenocarcinoma cell model, PC-3M model, or any other prostate cancer model can be used to test the methods and compositions disclosed herein.

[0345] Alternatively, or in addition, immunotherapy can be tested in human subjects, and known ___ can be used to monitor efficacy. Such methods can include, for example, steps of 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 cough, chest pain, weight loss, etc.). Methods for assessing the efficacy of cancer immunotherapy 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 these references is hereby incorporated by reference in its entirety for all purposes. VII. Kits

[0346] Also provided are kits containing reagents utilized in performing the methods disclosed herein, or kits containing the compositions, tools or instruments disclosed herein.

[0347] For example, such kits can include the lyophilized recombinant bacteria or Listeria strains disclosed herein, the immunogenic compositions disclosed herein, the pharmaceutical compositions disclosed herein, or the vaccines disclosed herein. Such kits can also include a solvent or diluent for reconstituting the lyophilized recombinant bacteria or Listeria strains. Additionally, such kits can further include instructional materials for the use of the lyophilized recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines for performing the methods disclosed herein. Such kits can further include an applicator, if desired. Model kits are described below, but the contents of other useful kits will be apparent in light of the present disclosure.

[0348] All of the above or below patent applications, websites, other published literature, accession numbers, etc. are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Even if different versions of a sequence are associated with an accession number at different times, the version associated with that accession number at the effective filing date of the present application is intended. This effective filing date means, where applicable, the earlier of the actual filing date or the filing date of the priority application, with reference to the accession number. Similarly, even if different versions of published literature, websites, etc. were published at different times, unless otherwise indicated, the version published closest to the effective filing date of the present application is intended. Unless otherwise specifically indicated, any feature, step, element, embodiment or aspect of the present invention can be used in combination with any other. The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, but 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 containing a Listeria strain, comprising: (a) providing a composition containing a Listeria strain in a formulation containing a buffer and sucrose; (b) cooling the composition provided in step (a) at a holding temperature 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 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 between about -5°C and about 25°C in a secondary drying step, thereby producing a lyophilized composition.

[0351] 2. The method according to 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 according to 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 according to any of the above embodiments, wherein the Listeria strain used in the composition in step (a) is a frozen Listeria strain thawed prior to step (a).

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

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

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

[0357] 8. The method according to embodiment 7, wherein the frozen Listeria strain is thawed at about 32°C and about 37°C.

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

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

[0360] 11. The method according to any one of embodiments 4 to 10, wherein the frozen Listeria strain is maintained at about 2°C to about 8°C for 24 hours or less after thawing.

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

[0362] 13. The method according to any of the above embodiments, wherein the buffer is a phosphate buffer.

[0363] 14. The method according to any of the above embodiments, wherein the formulation contains about 1% to about 5% w / v sucrose.

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

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

[0366] 17. The formulation contains about 1×10 per milliliter 9 ~ about 1×10 10The method according to any of the foregoing embodiments, comprising Listeria of colony forming units (CFU).

[0367] 18. The method according to any of the foregoing embodiments, wherein the formulation does not contain one or more of trehalose, monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

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

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

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

[0371] 22. The method according to any of the foregoing embodiments, wherein the freezing step (b) includes reducing the temperature to the holding temperature at a rate of about 1°C per minute.

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

[0373] 24. The method according to any of the foregoing embodiments, wherein the cooling in the freezing step (b) includes holding the composition at the holding temperature for about 2 hours.

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

[0375] 26. The method according to 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 according to embodiment 26, wherein the holding temperature in the primary drying step (c) is about -18°C.

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

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

[0379] 30. The method according to any of the preceding embodiments, wherein the end of the primary drying step (c) is about 12 to about 16 hours after the composition reaches the holding temperature.

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

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

[0382] 33. The method according to 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 according to embodiment 33, wherein the holding temperature in the secondary drying step (d) is about 0°C.

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

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

[0386] 37. The method according to any of the embodiments, 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 according to 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 according to any of the embodiments, wherein the secondary drying step (d) is at a vacuum pressure of about 0.09 mbar.

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

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

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

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

[0393] 44. The method according to any of the embodiments, wherein the lyophilized composition exhibits a survival rate of at least about 60% after being stored at about -20°C to about 4°C for about 12 months.

[0394] 45. The method according to embodiment 44, wherein the lyophilized composition exhibits a survival rate of at least about 75% after being stored at about -20°C to about 4°C for about 12 months.

[0395] 46. The method according to embodiment 45, wherein the lyophilized composition exhibits a survival rate of at least about 80% after being stored at about -20°C to about 4°C for about 12 months.

[0396] 47. The method according to any of the preceding embodiments, 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, the formulation contains about 1×10 9 ~about 1×10 10 colony forming units (CFU) of Listeria per milliliter, 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, and the residual moisture in the freeze-dried composition is between about 2.5% and about 4%. The method according to any of the preceding embodiments.

[0398] 49. The Listeria strain used in the composition in step (a) is a frozen Listeria strain that is thawed prior to step (a), the concentration of the frozen Listeria strain to be thawed is between about 1×10 9 ~about 1×10 10 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 held at about 2°C to about 8°C for 24 hours or less after thawing. The method according to 48.

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

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

[0401] 52. The method according to embodiment 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 in an episomal plasmid, 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.

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

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

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

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

[0406] 57. The formulation according to any one of embodiments 53 - 56, comprising about 1% - about 5% w / v sucrose.

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

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

[0409] 60. The formulation according to any one of embodiments 53 to 59, not containing one or more of trehalose, sodium glutamate (MSG) and recombinant human serum albumin (rHSA).

[0410] 61. The formulation according to embodiment 60, not containing trehalose, MSG or rHSA.

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

[0412] 63. The formulation according to any one of embodiments 53 to 62, wherein 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.

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

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

[0415] 66. The recombinant Listeria strain is an attenuated Listeria monocytogenes strain containing a deletion or inactivating mutation in actA, dal, and dat, the nucleic acid is in 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. The formulation according to embodiment 64.

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

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

[0418] 69. The lyophilized composition according to 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 according to embodiment 69, which does not contain trehalose, MSG, or rHSA.

[0420] 71. The lyophilized composition according to any one of embodiments 67 - 70, wherein the residual moisture in the lyophilized composition is between about 1% and about 5%.

[0421] 72. The lyophilized composition according to embodiment 71, wherein the residual moisture in the lyophilized composition is between about 2% and about 4%.

[0422] 73. The lyophilized composition according to any one of embodiments 67 - 72, wherein the residual moisture in the lyophilized composition is at least about 2.5%.

[0423] 74. The lyophilized composition according to embodiment 73, wherein the residual moisture in the lyophilized composition is at least about 3%.

[0424] 75. A lyophilized composition containing a Listeria strain, wherein the residual moisture in the lyophilized composition is at least about 2.5%.

[0425] 76. The lyophilized composition according to any one of embodiments 67 to 75, which shows a survival rate of at least about 60% after storage at about -20°C to about 4°C for about 12 months.

[0426] 77. The lyophilized composition according to embodiment 76, which shows a survival rate of at least about 75% after storage at about -20°C to about 4°C for about 12 months.

[0427] 78. The lyophilized composition according to embodiment 77, which shows a survival rate of at least about 80% after storage at about -20°C to about 4°C for about 12 months.

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

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

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

[0431] 82. The lyophilized composition according to embodiment 81, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain containing a deletion or inactivating mutation in prfA, the nucleic acid is in an episomal plasmid, and contains a second open reading frame encoding a D133V PrfA mutant protein.

[0432] 83. The lyophilized composition according to embodiment 81, 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 in an episomal plasmid, contains a second open reading frame encoding an alanine racemase enzyme or a D - amino acid aminotransferase enzyme, and the PEST - containing peptide is the N - terminal fragment of LLO.

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

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

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

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

[0437] 88. The method according to any one of embodiments 84 - 87, wherein the frozen Listeria strain is maintained at about 2°C to about 8°C for 24 hours or less after thawing.

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

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

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

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

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

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

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

[0445] 96. The method according to any one of embodiments 89 - 95, wherein the Listeria strain is a recombinant Listeria monocytogenes strain, the formulation comprises from about 2% to about 3% w / v sucrose, and the formulation does not contain 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 lyophilized composition containing Listeria strains, comprising: (a) providing a composition containing Listeria strains in a formulation containing a buffer and sucrose; (b) cooling the composition provided in step (a) in a freezing step, where the temperature is between about -32°C and -80°C if necessary; (c) exposing the composition produced in step (b) to a vacuum in a primary drying step, where the temperature is between about -10°C and -30°C if necessary; and (d) exposing the composition produced in step (c) to a vacuum in a secondary drying step, where the temperature is between about 5°C and 25°C if necessary and between about 5°C and 20°C if necessary, thereby producing a lyophilized composition.

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

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

[0450] 4. T...

Claims

1. A method for preparing a frozen Listeria strain for lyophilization, comprising: thawing the frozen Listeria strain at a temperature between 20° C. and 37° C. Including, the frozen Listeria strain is thawed in a formulation comprising a buffer and sucrose; the formulation comprises 1% to 5% w / v sucrose; The formulation does not contain trehalose, MSG or rHSA. method.

2. The method of claim 1, wherein the temperature is between 32°C and 37°C.

3. The method described in claim 2, wherein the temperature is 37°C.

4. The method of claim 1, wherein the frozen Listeria strain is thawed for 8 hours or less.

5. The method of claim 1, wherein the frozen Listeria strain is kept at 2°C to 8°C for no more than 24 hours after thawing.

6. The method of claim 1, wherein the formulation contains 2% to 3% w / v sucrose.

7. The method of claim 6, wherein the formulation contains 2.5% w / v sucrose.

8. The method of any one of claims 1 to 7, wherein the Listeria strain is a recombinant Listeria monocytogenes strain.

9. The method of claim 1, wherein the Listeria strain is a recombinant Listeria monocytogenes strain. the formulation comprises 2% to 3% w / v sucrose; The formulation does not contain trehalose, MSG or rHSA. The method according to any one of claims 1 to 8.

Images