TREA

Bacterial extracellular vesicles

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Information

  • Patent Grant
  • 12109243
  • Patent Number
    12,109,243
  • Date Filed
    Friday, December 2, 2022
    a year ago
  • Date Issued
    Tuesday, October 8, 2024
    a month ago
  • CPC
  • Field of Search
    • US
    • NON E00000
  • International Classifications
    • A61K35/74
    • A61P35/00
    • Disclaimer
      This patent is subject to a terminal disclaimer.
      Term Extension
      0
Information
Abstract
Provided herein are methods and compositions related to EVs useful as therapeutic agents.
Description
SUMMARY

In certain aspects, provided herein are pharmaceutical compositions comprising bacterial extracellular vesicles (EVs) useful for the treatment and/or prevention of disease (e.g., cancer, autoimmune disease, inflammatory disease, metabolic disease), as well as methods of making and/or identifying such EVs, and methods of using such pharmaceutical compositions (e.g., for the treatment of cancers, autoimmune diseases, inflammatory diseases, metabolic diseases, either alone or in combination with other therapeutics). In some embodiments, the pharmaceutical compositions comprise both EVs and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In certain embodiments, provided herein are pharmaceutical compositions comprising bacteria in the absence of EVs. In some embodiments, the pharmaceutical compositions comprise EVs in the absence of bacteria. In some embodiments, the pharmaceutical compositions comprise EVs and/or bacteria from one or more of the bacteria strains or species listed in Table 1 and/or Table 2.


In certain embodiments, the pharmaceutical composition comprises a specific ratio of bacteria to EV particles. For example, in some embodiments, the pharmaceutical composition comprises at least 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles. In some embodiments, the pharmaceutical composition comprises about 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles. In some embodiments, the pharmaceutical composition comprises no more than 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles. In some embodiments, the pharmaceutical composition comprises at least 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium. In some embodiments, the pharmaceutical composition comprises about 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium. In some embodiments, the pharmaceutical composition comprises no more than 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium.


In certain aspects, the EVs are from an engineered bacteria that is modified to enhance certain desirable properties. For example, in some embodiments, the engineered bacteria are modified to increase production of EVs. In some embodiments, the engineered bacteria are modified to produce EVs with enhanced oral delivery (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g. M-cells, goblet cells, enterocytes, dendritic cells, macrophages) to improve bioavailability systemically or in an appropriate niche (e.g., mesenteric lymph nodes, Peyer's patches, lamina propria, tumor draining lymph nodes, and/or blood), to enhance the immunomodulatory and/or therapeutic effect of the EVs they produce (e.g., either alone or in combination with another therapeutic agent), to enhance immune activation by the EVs they produce and/or to improve bacterial and/or EV manufacturing (e.g., greater stability, improved freeze-thaw tolerance, shorter generation times). In some embodiments, provided herein are methods of making such EVs and bacteria.


In certain embodiments, provided herein are methods of treating a subject who has cancer comprising administering to the subject a pharmaceutical composition described herein. In certain embodiments, provided herein are methods of treating a subject who has an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy) comprising administering to the subject a pharmaceutical composition described herein. In certain embodiments, provided herein are methods of treating a subject who has a metabolic disease comprising administering to the subject a pharmaceutical composition described herein.


In some embodiments, the method further comprises administering to the subject an antibiotic. In some embodiments, the method further comprises administering to the subject one or more other cancer therapies (e.g., surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, and/or the administration of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, and/or an adjuvant). In some embodiments, the method further comprises the administration of another therapeutic bacterium and/or EV. In some embodiments, the method further comprises the administration of an immune suppressant and/or an anti-inflammatory agent. In some embodiments, the method further comprises the administration of a metabolic disease therapeutic agent.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 shows the efficacy of iv administered Blautia massiliensis compared to that of intraperitoneally (i.p.) administered anti-PD-1 in a mouse colorectal carcinoma model.



FIG. 2 shows inhibition of tumor growth (by volume) by the iv administration of Blautia massiliensis compared to intraperitoneally (i.p.) administered anti-PD-1 in a mouse colorectal carcinoma model.



FIG. 3A shows the efficacy of orally or intra-peritoneally administered Prevotella histicola and P. histicola-derived EVs in reducing antigen-specific ear swelling (ear thickness) 24 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model. Efficacy was seen in both the oral and i.p. administration groups.



FIG. 3B shows the efficacy of orally or intra-peritoneally administered Prevotella histicola and P. histicola-derived EVs in reducing antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model.



FIG. 3C shows the efficacy of intra-peritoneally administered P. histicola-derived EVs at the dose indicated (10 μg, 3 μg, 1 μg, and 0.1 μg) in reducing antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model.



FIG. 3D shows the ability of intra-peritoneally administered Prevotella histicola-derived EVs to reduce expression of IL-1ß 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model.



FIG. 3E shows the ability of intra-peritoneally administered Prevotella histicola-derived EVs to increase accumulation of Tregs in the cervical lymph nodes 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model.



FIG. 4 shows that P. histicola was efficacious at reducing the NASH activity score (NAS) in mice receiving a methionine choline deficient (MCD) diet, which induces NASH symptoms.



FIG. 5A shows that P. histicola reduced steatosis in mice that were fed an MCD diet.



FIG. 5B and FIG. 5C show that P. histicola reduced inflammation in mice that were fed an MCD diet.



FIG. 5D shows that P. histicola reduced ballooning in mice that were fed an MCD diet.



FIG. 6 shows that P. histicola reduced hepatic total cholesterol in mice that were fed an MCD diet.



FIG. 7A and FIG. 7B show that P. histicola reduced the fibrosis score in mice that were fed an MCD diet.



FIG. 8A shows the efficacy of administering Prevotella histicola, P. melanogenica, P. histicola-derived EVs, P. melanogenica-derived EVs in reducing antigen-specific ear swelling (ear thickness) 24 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model. P. melanogenica-derived EVs is more effective than P. melanogenica.



FIG. 8B shows the efficacy of administering Prevotella histicola, P. melanogenica, P. histicola-derived EVs, P. melanogenica-derived EVs in reducing antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model. P. melanogenica-derived EVs is more effective than P. melanogenica.



FIG. 9A shows the effect of P. histicola on hepatic free fatty acids in mice that were fed an MCD diet.



FIG. 9B shows the effect of P. histicola on hepatic total cholesterol in mice that were fed an MCD diet.



FIG. 9C shows the effect of P. histicola on hepatic triglycerides in mice that were fed an MCD diet.



FIG. 9D shows the effect of P. histicola and P. melanogenicaon alanine aminotransferase in mice that were fed an MCD diet.



FIG. 9E shows the effect of P. histicola and P. melanogenica on aspartate aminotransferase in mice that were fed an MCD diet.



FIG. 10A shows that P. histicola is efficacious at reducing the NASH activity score (NAS) in mice receiving a methionine choline deficient (MCD) diet, which induces NASH symptoms.



FIG. 10B shows that Prevotella histicola, P. melanogenica, P. histicola in combination with OCD at reducing the NASH activity score (NAS) in mice receiving a methionine choline deficient (MCD) diet, which induces NASH symptoms.



FIG. 11 shows the efficacy of orally administered Veillonella tobetsuensis and Veillonella parvula Strains in reducing antigen-specific ear swelling (ear thickness) at 24 hours compared to vehicle (negative control), anti-inflammatory Dexamethasone (positive control), and Bifidobacterium animalis lactis in a KLH-based delayed type hypersensitivity mouse model.



FIG. 12 shows the efficacy of EVs from two Veillonella tobetsuensis and Veillonella parvula strains compared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model.



FIG. 13 shows the efficacy of EVs from Veillonella tobetsuensis and Veillonella parvula Strains compared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day 11.



FIG. 14 shows the dose and route of administration dependent efficacy of EVs from Veillonella tobetsuensis and Veillonella parvula strains compared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model.



FIG. 15 shows the dose and route of administration dependent efficacy of EVs from Veillonella tobetsuensis and Veillonella parvula strains compared to intraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day 11.



FIG. 16 shows that Veillonella parvula was efficacious at reducing the NASH activity score (NAS) in mice receiving a methionine choline deficient (MCD) diet, which induces NASH symptoms.



FIG. 17 shows that Veillonella parvula reduced Fibrosis in mice that were fed an MCD diet.



FIG. 18 shows that Veillonella parvula reduced Hepatic Total Cholesterol in mice that were fed an MCD diet.



FIG. 19 shows that Veillonella parvula reduced Hepatic Triglycerides in mice that were fed an MCD diet



FIG. 20 shows the efficacy of Burkholderia pseudomallei EVs compared to intravenously (i.v.) administered anti-PD-1 or vehicle in a mouse colorectal carcinoma model.



FIG. 21 shows the efficacy of Burkholderia pseudomallei EVs compared to intravenously (i.v.) administered anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day 11. ** The difference in tumor volume in the Burkholderia pseudomallei EV treated group compared to vehicle control group on day 11 was highly significant, with a P-value of 0.0011 as determined by T-test (two-tailed, unpaired, welch-corrected) calculated in GraphPad.



FIG. 22 shows the efficacy of Neisseria Meningitidis EVs compared to that of intraperitoneally (i.p.) administered anti-PD-1 or vehicle in a mouse colorectal carcinoma model.



FIG. 23 shows the efficacy of Neisseria Meningitidis EVs compared to that of intraperitoneally (i.p.) administered anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day 11.





DETAILED DESCRIPTION
Definitions

“Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.


“Administration” broadly refers to a route of administration of a composition to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration. The pharmaceutical compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In preferred embodiments, the pharmaceutical compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.


As used herein, the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.


The terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include Fab, Fab′, F(ab′)2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.


“Cancer” broadly refers to an uncontrolled, abnormal growth of a host's own cells leading to invasion of surrounding tissue and potentially tissue distal to the initial site of abnormal cell growth in the host. Major classes include carcinomas which are cancers of the epithelial tissue (e.g., skin, squamous cells); sarcomas which are cancers of the connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias which are cancers of blood forming tissue (e.g., bone marrow tissue); lymphomas and myelomas which are cancers of immune cells; and central nervous system cancers which include cancers from brain and spinal tissue. “Cancer(s),” “neoplasm(s),” and “tumor(s)” are used herein interchangeably. As used herein, “cancer” refers to all types of cancer or neoplasm or malignant tumors including leukemias, carcinomas and sarcomas, whether new or recurring. Specific examples of cancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed type tumors. Non-limiting examples of cancers are new or recurring cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus and medulloblastoma.


“Cellular augmentation” broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself. Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells. Environments of particular interest are the microenvironments where cancer cells reside or locate. In some instances, the microenvironment is a tumor microenvironment or a tumor draining lymph node. In other instances, the microenvironment is a pre-cancerous tissue site or the site of local administration of a composition or a site where the composition will accumulate after remote administration.


“Clade” refers to the OTUs or members of a phylogenetic tree that are downstream of a statistically valid node in a phylogenetic tree. The clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity. “Operational taxonomic units,” “OTU” (or plural, “OTUs”) refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. In 16S embodiments, OTUs that share ≥97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU (see e.g. Claesson M J. Wang Q, O'Sullivan O, Greene-Diniz R. Cole J R. Ros R P. and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361. 1929-1940.) In embodiments involving the complete genome, MLSTs, specific genes, or sets of genes OTUs that share ≥95% average nucleotide identity are considered the same OTU (see e.g. Achtman M. and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Such characterization employs, e.g., WGS data or a whole genome sequence.


A “combination” of EVs from two or more microbial strains includes the physical co-existence of the two EVs, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the EVs from the two strains.


The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state.


As used herein, the term “Dysbiosis” refers to a state in which the synergy between microbes and the tumor is broken such as the microbes no longer support the nucleation, maintenance, progression or spread or metastasis of a tumor.


The term “epitope” means a protein determinant capable of specific binding to an antibody or T cell receptor. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.


As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human intervention and the progeny of any such bacteria. Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.


The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.


“Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).


As used herein, the term “immune disorder” refers to any disease, disorder or disease symptom caused by an activity of the immune system, including autoimmune diseases, inflammatory diseases and allergies. Immune disorders include, but are not limited to, autoimmune diseases (e.g., Lupus, Scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave's disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, pernicious anemia and/or myopathy), inflammatory diseases (e.g., acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and/or interstitial cystitis), and/or an allergies (e.g., food allergies, drug allergies and/or environmental allergies).


“Immunotherapy” is treatment that uses a subject's immune system to treat disease (e.g., immune disease, inflammatory disease, metabolic disease, cancer) and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.


The term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}3 fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold, 10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 fold greater after treatment when compared to a pre-treatment state. Properties that may be increased include immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites, and cytokines.


“Innate immune agonists” or “immuno-adjuvants” are small molecules, proteins, or other agents that specifically target innate immune receptors including Toll-Like Receptors (TLR), NOD receptors, RLRs, C-type lectin receptors, STING-cGAS Pathway components, inflammasome complexes. For example, LPS is a TLR-4 agonist that is bacterially derived or synthesized and aluminum can be used as an immune stimulating adjuvant. immuno-adjuvants are a specific class of broader adjuvant or adjuvant therapy. Examples of STING agonists include, but are not limited to, 2′3′-cGAMP, 3′3′-cGAMP, c-di-AMP, c-di-GMP, 2′2′-cGAMP, and 2′3′-cGAM(PS)2 (Rp/Sp) (Rp, Sp-isomers of the bis-phosphorothioate analog of 2′3′-cGAMP). Examples of TLR agonists include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO and TLRI 1. Examples of NOD agonists include, but are not limited to, N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide (MDP)), gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), and desmuramylpeptides (DMP).


The “internal transcribed spacer” or “ITS” is a piece of non-functional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi. The rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively. These two intercistronic segments between the 18S and 5.8S and 5.8S and 28S regions are removed by splicing and contain significant variation between species for barcoding purposes as previously described (Schoch et al Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used for phylogenetic reconstruction however the ITS can serve this function as it is generally highly conserved but contains hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most fungus.


The term “isolated” or “enriched” encompasses a microbe, EV or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of microbial compositions provided herein, the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions and the microbial components thereof are generally purified from residual habitat products.”


“Metabolite” as used herein refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.


“Microbe” refers to any natural or engineered organism characterized as a bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism. Examples of gut microbes include: Actinomyces graevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorea longicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothia mucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.


“Microbiome” broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses. Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner. The microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome. The microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state (e.g., precancerous or cancerous state) or treatment conditions (e.g., antibiotic treatment, exposure to different microbes). In some aspects, the microbiome occurs at a mucosal surface. In some aspects, the microbiome is a gut microbiome. In some aspects, the microbiome is a tumor microbiome.


A “microbiome profile” or a “microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome. In some embodiments, a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome. In some embodiments, a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more cancer-associated bacterial strains are present in a sample. In some embodiments, the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample. In some embodiments, the microbiome profile is a cancer-associated microbiome profile. A cancer-associated microbiome profile is a microbiome profile that occurs with greater frequency in a subject who has cancer than in the general population. In some embodiments, the cancer-associated microbiome profile comprises a greater number of or amount of cancer-associated bacteria than is normally present in a microbiome of an otherwise equivalent tissue or sample taken from an individual who does not have cancer.


“Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form. Examples of bacterial modifications include genetic modification, gene expression, phenotype modification, formulation, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity. Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium that increase or decrease virulence.


As used herein, a gene is “overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions. Similarly, a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.


The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.


An “oncobiome” as used herein comprises pathogenic, tumorigenic and/or cancer-associated microbiota, wherein the microbiota comprises one or more of a virus, a bacterium, a fungus, a protist, a parasite, or another microbe.


“Oncotrophic” or “oncophilic” microbes and bacteria are microbes that are highly associated or present in a cancer microenvironment. They may be preferentially selected for within the environment, preferentially grow in a cancer microenvironment or hone to a said environment.


“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. For 16S, OTUs that share ≥97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share ≥95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms.


Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein.


As used herein, the term “extracellular vesicle” or “EV” or refers to a composition derived from a bacteria that comprises bacterial lipids, and bacterial proteins and/or bacterial nucleic acids and/or carbohydrate moieties contained in a nanoparticle. These EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different lipid species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different protein species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different nucleic acid species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different carbohydrate species.


As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a EV or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. An EV may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.” In some embodiments, purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. EV compositions and the microbial components thereof are, e.g., purified from residual habitat products.


As used herein, the term “purified EV composition” or “EV composition” refer to a preparation that includes EVs that have been separated from at least one associated substance found in a source material (e.g. separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments the EVs are concentrated by 2 fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000 fold.


“Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject. For example, microbes live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community). Substantially free of residual habitat products means that the microbial composition no longer contains the biological matter associated with the microbial environment on or in the human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community. Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms. Substantially free of residual habitat products may also mean that the microbial composition contains no detectable cells from a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including microbial viruses (e.g., phage)), fungal, mycoplasmal contaminants. In another embodiment, it means that fewer than 1×10-2%, 1×10-3%, 1×10-4%, 1×10-5%, 1×10-6%, 1×10-7%, 1×10-8% of the viable cells in the microbial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting. Thus, contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology. Alternatively, reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10−8 or 10−9), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior. Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.


As used herein, “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner. Typically, an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10−7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g., BSA, casein). Alternatively, specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.


The terms “subject” or “patient” refers to any animal. A subject or a patient described as “in need thereof” refers to one in need of a treatment for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).


“Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.


As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening. Thus, in one embodiment, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.


Bacteria


In certain aspects, provided herein are pharmaceutical compositions that comprise bacteria and/or EVs made from bacteria.


In some embodiments, the bacteria from which the EVs are obtained are modified to enhance EV production, to enhance oral delivery of the produced EVs (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g. M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the produced EVs (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the produced EVs (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins). In some embodiments, the engineered bacteria described herein are modified to improve bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times). For example, in some embodiments, the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes. The engineered microbe(s) may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.


As used herein, the term “bacteria” broadly refers to the domain of prokaryotic organisms, including Gram positive and Gram negative organisms. Examples of species and/or strains of bacteria that can be used to produce the EVs described herein are provided in Tables 1 and/or Table 2 and elsewhere throughout the specification. In some embodiments, the bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed in Table 1 and/or Table 2. In some embodiments, the EVs are from an oncotrophic bacteria. In some embodiments, the EVs are from an immunostimulatory bacteria. In some embodiments the EVs are from an immunosuppressive bacteria. In some embodiments, the EVs are from an immunomodulatory bacteria. In certain embodiments, EVs are generated from a combination of bacterial strains provided herein. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains. In some embodiments combination includes EVs from bacterial strains listed in Table 1 and/or Table 2 and/or bacterial strains having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed in Table 1 and/or Table 2.









TABLE 1







Exemplary Bacterial Strains








OTU
Public DB Accession






Abiotrophia
defectiva

ACIN02000016



Abiotrophia para_adiacens
AB022027



Abiotrophia sp. oral clone P4PA_155 P1
AY207063



Acetanaerobacterium
elongatum

NR_042930



Acetivibrio
cellulolyticus

NR_025917



Acetivibrio
ethanolgignens

FR749897



Acetobacter
aceti

NR_026121



Acetobacter
fabarum

NR_042678



Acetobacter
lovaniensis

NR_040832



Acetobacter
malorum

NR_025513



Acetobacter
orientalis

NR_028625



Acetobacter
pasteurianus

NR_026107



Acetobacter
pomorum

NR_042112



Acetobacter
syzygii

NR_040868



Acetobacter
tropicalis

NR_036881



Acetobacteraceae
bacterium AT_5844
AGEZ01000040



Acholeplasma
laidlawii

NR_074448



Achromobacter
denitrificans

NR_042021



Achromobacter
piechaudii

ADMS01000149



Achromobacter
xylosoxidans

ACRC01000072



Acidaminococcus
fermentans

CP001859



Acidaminococcus
intestini

CP003058



Acidaminococcus sp. D21
ACGB01000071



Acidilobus
saccharovorans

AY350586



Acidithiobacillus
ferrivorans

NR_074660



Acidovorax sp. 98_63833
AY258065



Acinetobacter
baumannii

ACYQ01000014



Acinetobacter
calcoaceticus

AM157426



Acinetobacter genomosp. C1
AY278636



Acinetobacter
haemolyticus

ADMT01000017



Acinetobacter
johnsonii

ACPL01000162



Acinetobacter
junii

ACPM01000135



Acinetobacter
lwoffii

ACPN01000204



Acinetobacter
parvus

AIEB01000124



Acinetobacter
radioresistens

ACVR01000010



Acinetobacter
schindleri

NR_025412



Acinetobacter sp. 56A1
GQ178049



Acinetobacter sp. CIP 101934
JQ638573



Acinetobacter sp. CIP 102143
JQ638578



Acinetobacter sp. CIP 53.82
JQ638584



Acinetobacter sp. M16_22
HM366447



Acinetobacter sp. RUH2624
ACQF01000094



Acinetobacter sp. SH024
ADCH01000068



Actinobacillus
actinomycetemcomitans

AY362885



Actinobacillus
minor

ACFT01000025



Actinobacillus
pleuropneumoniae

NR_074857



Actinobacillus
succinogenes

CP000746



Actinobacillus
ureae

AEVG01000167



Actinobaculum
massiliae

AF487679



Actinobaculum
schaalii

AY957507



Actinobaculum sp. BM#101342
AY282578



Actinobaculum sp. P2P_19 P1
AY207066



Actinomyces
cardiffensis

GU470888



Actinomyces
europaeus

NR_026363



Actinomyces
funkei

HQ906497



Actinomyces genomosp. C1
AY278610



Actinomyces genomosp. C2
AY278611



Actinomyces genomosp. P1 oral clone MB6_C03
DQ003632



Actinomyces
georgiae

GU561319



Actinomyces
israelii

AF479270



Actinomyces
massiliensis

AB545934



Actinomyces
meyeri

GU561321



Actinomyces
naeslundii

X81062



Actinomyces
nasicola

AJ508455



Actinomyces
neuii

X71862



Actinomyces
odontolyticus

ACYT01000123



Actinomyces
oricola

NR_025559



Actinomyces
orihominis

AJ575186



Actinomyces
oris

BABV01000070



Actinomyces sp. 7400942
EU484334



Actinomyces sp. c109
AB167239



Actinomyces sp. CCUG 37290
AJ234058



Actinomyces sp. ChDC B197
AF543275



Actinomyces sp. GEJ15
GU561313



Actinomyces sp. HKU31
HQ335393



Actinomyces sp. ICM34
HQ616391



Actinomyces sp. ICM41
HQ616392



Actinomyces sp. ICM47
HQ616395



Actinomyces sp. ICM54
HQ616398



Actinomyces sp. M2231_94_1
AJ234063



Actinomyces sp. oral clone GU009
AY349361



Actinomyces sp. oral clone GU067
AY349362



Actinomyces sp. oral clone IO076
AY349363



Actinomyces sp. oral clone IO077
AY349364



Actinomyces sp. oral clone IP073
AY349365



Actinomyces sp. oral clone IP081
AY349366



Actinomyces sp. oral clone JA063
AY349367



Actinomyces sp. oral taxon 170
AFBL01000010



Actinomyces sp. oral taxon 171
AECW01000034



Actinomyces sp. oral taxon 178
AEUH01000060



Actinomyces sp. oral taxon 180
AEPP01000041



Actinomyces sp. oral taxon 848
ACUY01000072



Actinomyces sp. oral taxon C55
HM099646



Actinomyces sp. TeJ5
GU561315



Actinomyces
urogenitalis

ACFH01000038



Actinomyces
viscosus

ACRE01000096



Adlercreutzia
equolifaciens

AB306661



Aerococcus
sanguinicola

AY837833



Aerococcus
urinae

CP002512



Aerococcus
urinaeequi

NR_043443



Aerococcus
viridans

ADNT01000041



Aeromicrobium
marinum

NR_025681



Aeromicrobium sp. JC14
JF824798



Aeromonas
allosaccharophila

S39232



Aeromonas
enteropelogenes

X71121



Aeromonas
hydrophila

NC_008570



Aeromonas
jandaei

X60413



Aeromonas
salmonicida

NC_009348



Aeromonas
trota

X60415



Aeromonas
veronii

NR_044845



Afipia genomosp. 4
EU117385



Aggregatibacter
actinomycetemcomitans

CP001733



Aggregatibacter
aphrophilus

CP001607


Aggregatibacter segnis
AEPS01000017



Agrobacterium
radiobacter

CP000628



Agrobacterium
tumefaciens

AJ389893



Agrococcus
jenensis

NR_026275



Akkermansia
muciniphila

CP001071



Alcaligenes
faecalis

AB680368



Alcaligenes sp. CO14
DQ643040



Alcaligenes sp. S3
HQ262549



Alicyclobacillus
acidocaldarius

NR_074721



Alicyclobacillus
acidoterrestris

NR_040844



Alicyclobacillus
contaminans

NR_041475



Alicyclobacillus
cycloheptanicus

NR_024754



Alicyclobacillus
herbarius

NR_024753



Alicyclobacillus
pomorum

NR_024801



Alicyclobacillus sp. CCUG 53762
HE613268



Alistipes
finegoldii

NR_043064



Alistipes
indistinctus

AB490804



Alistipes
onderdonkii

NR_043318



Alistipes
putredinis

ABFK02000017



Alistipes
shahii

FP929032



Alistipes sp. HGB5
AENZ01000082



Alistipes sp. JC50
JF824804



Alistipes sp. RMA 9912
GQ140629



Alkaliphilus
metalliredigenes

AY137848



Alkaliphilus
oremlandii

NR_043674



Alloscardovia
omnicolens

NR_042583



Alloscardovia sp. OB7196
AB425070



Anaerobaculum
hydrogeniformans

ACJX02000009



Anaerobiospirillum
succiniciproducens

NR_026075



Anaerobiospirillum
thomasii

AJ420985



Anaerococcus
hydrogenalis

ABXA01000039



Anaerococcus
lactolyticus

ABYO01000217



Anaerococcus
octavius

NR_026360



Anaerococcus
prevotii

CP001708



Anaerococcus sp. 8404299
HM587318



Anaerococcus sp. 8405254
HM587319



Anaerococcus sp. 9401487
HM587322



Anaerococcus sp. 9403502
HM587325



Anaerococcus sp. gpac104
AM176528



Anaerococcus sp. gpac126
AM176530



Anaerococcus sp. gpac155
AM176536



Anaerococcus sp. gpac199
AM176539



Anaerococcus sp. gpac215
AM176540



Anaerococcus
tetradius

ACGC01000107



Anaerococcus
vaginalis

ACXU01000016



Anaerofustis
stercorihominis

ABIL02000005



Anaeroglobus
geminatus

AGCJ01000054



Anaerosporobacter
mobilis

NR_042953



Anaerostipes
caccae

ABAX03000023



Anaerostipes sp. 3_2_56FAA
ACWB01000002



Anaerotruncus
colihominis

ABGD02000021



Anaplasma
marginale

ABOR01000019



Anaplasma
phagocytophilum

NC_007797



Aneurinibacillus
aneurinilyticus

AB101592



Aneurinibacillus
danicus

NR_028657



Aneurinibacillus
migulanus

NR_036799



Aneurinibacillus
terranovensis

NR_042271



Aneurinibacillus
thermoaerophilus

NR_029303



Anoxybacillus
contaminans

NR_029006



Anoxybacillus
flavithermus

NR_074667



Arcanobacterium
haemolyticum

NR_025347



Arcanobacterium
pyogenes

GU585578



Arcobacter
butzleri

AEPT01000071



Arcobacter
cryaerophilus

NR_025905



Arthrobacter
agilis

NR_026198



Arthrobacter
arilaitensis

NR_074608



Arthrobacter
bergerei

NR_025612



Arthrobacter
globiformis

NR_026187



Arthrobacter
nicotianae

NR_026190



Atopobium
minutum

HM007583



Atopobium
parvulum

CP001721



Atopobium
rimae

ACFE01000007



Atopobium sp. BS2
HQ616367



Atopobium sp. F0209
EU592966



Atopobium sp. ICM42b10
HQ616393



Atopobium sp. ICM57
HQ616400



Atopobium
vaginae

AEDQ01000024



Aurantimonas
coralicida

AY065627



Aureimonas
altamirensis

FN658986



Auritibacter
ignavus

FN554542



Averyella
dalhousiensis

DQ481464



Bacillus
aeolius

NR_025557



Bacillus
aerophilus

NR_042339



Bacillus
aestuarii

GQ980243



Bacillus
alcalophilus

X76436



Bacillus
amyloliquefaciens

NR_075005



Bacillus
anthracis

AAEN01000020



Bacillus
atrophaeus

NR_075016



Bacillus
badius

NR_036893



Bacillus
cereus

ABDJ01000015



Bacillus
circulans

AB271747



Bacillus
clausii

FN397477



Bacillus
coagulans

DQ297928



Bacillus
firmus

NR_025842



Bacillus
flexus

NR_024691



Bacillus
fordii

NR_025786



Bacillus
gelatini

NR_025595



Bacillus
halmapalus

NR_026144



Bacillus
halodurans

AY144582



Bacillus
herbersteinensis

NR_042286



Bacillus
horti

NR_036860



Bacillus
idriensis

NR_043268



Bacillus
lentus

NR_040792



Bacillus
licheniformis

NC_006270



Bacillus
megaterium

GU252124



Bacillus
nealsonii

NR_044546



Bacillus
niabensis

NR_043334



Bacillus
niacini

NR_024695



Bacillus
pocheonensis

NR_041377



Bacillus
pumilus

NR_074977



Bacillus
safensis

JQ624766



Bacillus
simplex

NR_042136



Bacillus
sonorensis

NR_025130



Bacillus sp. 10403023 MM10403188
CAET01000089



Bacillus sp. 2_A_57_CT2
ACWD01000095



Bacillus sp. 2008724126
GU252108



Bacillus sp. 2008724139
GU252111



Bacillus sp. 7_16AIA
FN397518



Bacillus sp. 9_3AIA
FN397519



Bacillus sp. AP8
JX101689



Bacillus sp. B27(2008)
EU362173


Bacillus sp. BT1B_CT2
ACWC01000034



Bacillus sp. GB1.1
FJ897765



Bacillus sp. GB9
FJ897766



Bacillus sp. HU19.1
FJ897769



Bacillus sp. HU29
FJ897771



Bacillus sp. HU33.1
FJ897772



Bacillus sp. JC6
JF824800



Bacillus sp. oral taxon F26
HM099642



Bacillus sp. oral taxon F28
HM099650



Bacillus sp. oral taxon F79
HM099654



Bacillus sp. SRC_DSF1
GU797283



Bacillus sp. SRC_DSF10
GU797292



Bacillus sp. SRC_DSF2
GU797284



Bacillus sp. SRC_DSF6
GU797288



Bacillus sp. tc09
HQ844242



Bacillus sp. zh168
FJ851424



Bacillus
sphaericus

DQ286318



Bacillus
sporothermodurans

NR_026010



Bacillus
subtilis

EU627588



Bacillus
thermoamylovorans

NR_029151



Bacillus
thuringiensis

NC_008600



Bacillus
weihenstephanensis

NR_074926



Bacteroidales bacterium ph8
JN837494



Bacteroidales genomosp. P1
AY341819



Bacteroidales genomosp. P2 oral clone
DQ003613


MB1_G13




Bacteroidales genomosp. P3 oral clone
DQ003615


MB1_G34




Bacteroidales genomosp. P4 oral clone
DQ003617


MB2_G17




Bacteroidales genomosp. P5 oral clone
DQ003619


MB2_P04




Bacteroidales genomosp. P6 oral clone
DQ003634


MB3_C19




Bacteroidales genomosp. P7 oral clone
DQ003623


MB3_P19




Bacteroidales genomosp. P8 oral clone
DQ003626


MB4_G15




Bacteroides
acidifaciens

NR_028607



Bacteroides
barnesiae

NR_041446



Bacteroides
caccae

EU136686



Bacteroides
cellulosilyticus

ACCH01000108



Bacteroides
clarus

AFBM01000011



Bacteroides
coagulans

AB547639



Bacteroides
coprocola

ABIY02000050



Bacteroides
coprophilus

ACBW01000012



Bacteroides
dorei

ABWZ01000093



Bacteroides
eggerthii

ACWG01000065



Bacteroides
faecis

GQ496624



Bacteroides
finegoldii

AB222699



Bacteroides
fluxus

AFBN01000029



Bacteroides
fragilis

AP006841



Bacteroides
galacturonicus

DQ497994



Bacteroides
helcogenes

CP002352



Bacteroides
heparinolyticus

JN867284



Bacteroides
intestinalis

ABJL02000006



Bacteroides
massiliensis

AB200226



Bacteroides
nordii

NR_043017



Bacteroides
oleiciplenus

AB547644



Bacteroides
ovatus

ACWH01000036



Bacteroides
pectinophilus

ABVQ01000036



Bacteroides
plebeius

AB200218



Bacteroides
pyogenes

NR_041280



Bacteroides
salanitronis

CP002530



Bacteroides
salyersiae

EU136690



Bacteroides sp. 1_1_14
ACRP01000155



Bacteroides sp. 1_1_30
ADCL01000128



Bacteroides sp. 1_1_6
ACIC01000215



Bacteroides sp. 2_1_22
ACPQ01000117



Bacteroides sp. 2_1_56FAA
ACWI01000065



Bacteroides sp. 2_2_4
ABZZ01000168



Bacteroides sp. 20_3
ACRQ01000064



Bacteroides sp. 3_1_19
ADCJ01000062



Bacteroides sp. 3_1_23
ACRS01000081



Bacteroides sp. 3_1_33FAA
ACPS01000085



Bacteroides sp. 3_1_40A
ACRT01000136



Bacteroides sp. 3_2_5
ACIB01000079



Bacteroides sp. 315_5
FJ848547



Bacteroides sp. 31SF15
AJ583248



Bacteroides sp. 31SF18
AJ583249



Bacteroides sp. 35AE31
AJ583244



Bacteroides sp. 35AE37
AJ583245



Bacteroides sp. 35BE34
AJ583246



Bacteroides sp. 35BE35
AJ583247



Bacteroides sp. 4_1_36
ACTC01000133



Bacteroides sp. 4_3_47FAA
ACDR02000029



Bacteroides sp. 9_1_42FAA
ACAA01000096



Bacteroides sp. AR20
AF139524



Bacteroides sp. AR29
AF139525



Bacteroides sp. B2
EU722733



Bacteroides sp. D1
ACAB02000030



Bacteroides sp. D2
ACGA01000077



Bacteroides sp. D20
ACPT01000052



Bacteroides sp. D22
ADCK01000151



Bacteroides sp. F_4
AB470322



Bacteroides sp. NB_8
AB117565



Bacteroides sp. WH2
AY895180



Bacteroides sp. XB12B
AM230648



Bacteroides sp. XB44A
AM230649



Bacteroides
stercoris

ABFZ02000022



Bacteroides
thetaiotaomicron

NR_074277



Bacteroides
uniformis

AB050110



Bacteroides
ureolyticus

GQ167666



Bacteroides
vulgatus

CP000139



Bacteroides
xylanisolvens

ADKP01000087



Bacteroidetes bacterium oral taxon D27
HM099638



Bacteroidetes bacterium oral taxon F31
HM099643



Bacteroidetes bacterium oral taxon F44
HM099649



Barnesiella
intestinihominis

AB370251



Barnesiella
viscericola

NR_041508



Bartonella
bacilliformis

NC_008783


Bartonella grahamii
CP001562



Bartonella
henselae

NC_005956



Bartonella
quintana

BX897700



Bartonella
tamiae

EF672728



Bartonella
washoensis

FJ719017



Bdellovibrio sp. MPA
AY294215



Bifidobacteriaceae genomosp. C1
AY278612



Bifidobacterium
adolescentis

AAXD02000018



Bifidobacterium
angulatum

ABYS02000004



Bifidobacterium
animalis

CP001606



Bifidobacterium
bifidum

ABQP01000027



Bifidobacterium
breve

CP002743



Bifidobacterium
catenulatum

ABXY01000019



Bifidobacterium
dentium

CP001750



Bifidobacterium
gallicum

ABXB03000004



Bifidobacterium
infantis

AY151398



Bifidobacterium
kashiwanohense

AB491757



Bifidobacterium
longum

ABQQ01000041



Bifidobacterium
pseudocatenulatum

ABXX02000002



Bifidobacterium
pseudolongum

NR_043442



Bifidobacterium
scardovii

AJ307005



Bifidobacterium sp. HM2
AB425276



Bifidobacterium sp. HMLN12
JF519685



Bifidobacterium sp. M45
HM626176



Bifidobacterium sp. MSX5B
HQ616382



Bifidobacterium sp. TM_7
AB218972



Bifidobacterium
thermophilum

DQ340557



Bifidobacterium
urinalis

AJ278695



Bilophila
wadsworthia

ADCP01000166



Bisgaard Taxon
AY683487



Bisgaard Taxon
AY683489



Bisgaard Taxon
AY683491



Bisgaard Taxon
AY683492



Blastomonas
natatoria

NR_040824



Blautia
coccoides

AB571656



Blautia
glucerasea

AB588023



Blautia
glucerasei

AB439724



Blautia
hansenii

ABYU02000037



Blautia
hydrogenotrophica

ACBZ01000217



Blautia
luti

AB691576



Blautia
producta

AB600998



Blautia
schinkii

NR_026312



Blautia sp. M25
HM626178



Blautia
stercoris

HM626177



Blautia
wexlerae

EF036467



Bordetella
bronchiseptica

NR_025949



Bordetella
holmesii

AB683187



Bordetella
parapertussis

NR_025950



Bordetella
pertussis

BX640418



Borrelia
afzelii

ABCU01000001



Borrelia
burgdorferi

ABGI01000001



Borrelia
crocidurae

DQ057990



Borrelia
duttonii

NC_011229



Borrelia
garinii

ABJV01000001



Borrelia
hermsii

AY597657



Borrelia
hispanica

DQ057988



Borrelia
persica

HM161645



Borrelia
recurrentis

AF107367



Borrelia sp. NE49
AJ224142



Borrelia
spielmanii

ABKB01000002



Borrelia
turicatae

NC_008710



Borrelia
valaisiana

ABCY01000002



Brachybacterium
alimentarium

NR_026269



Brachybacterium
conglomeratum

AB537169



Brachybacterium
tyrofermentans

NR_026272



Brachyspira
aalborgi

FM178386



Brachyspira
pilosicoli

NR_075069



Brachyspira sp. HIS3
FM178387



Brachyspira sp. HIS4
FM178388



Brachyspira sp. HIS5
FM178389



Brevibacillus
agri

NR_040983



Brevibacillus
brevis

NR_041524



Brevibacillus
centrosporus

NR_043414



Brevibacillus
choshinensis

NR_040980



Brevibacillus
invocatus

NR_041836



Brevibacillus
laterosporus

NR_037005



Brevibacillus
parabrevis

NR_040981



Brevibacillus
reuszeri

NR_040982



Brevibacillus sp. phR
JN837488



Brevibacillus
thermoruber

NR_026514



Brevibacterium
aurantiacum

NR_044854



Brevibacterium
casei

JF951998



Brevibacterium
epidermidis

NR_029262



Brevibacterium
frigoritolerans

NR_042639



Brevibacterium
linens

AJ315491



Brevibacterium
mcbrellneri

ADNU01000076



Brevibacterium
paucivorans

EU086796



Brevibacterium
sanguinis

NR_028016



Brevibacterium sp. H15
AB177640



Brevibacterium sp. JC43
JF824806



Brevundimonas
subvibrioides

CP002102



Brucella
abortus

ACBJ01000075



Brucella
canis

NR_044652



Brucella
ceti

ACJD01000006



Brucella
melitensis

AE009462



Brucella
microti

NR_042549



Brucella
ovis

NC_009504



Brucella sp. 83_13
ACBQ01000040



Brucella sp. BO1
EU053207



Brucella
suis

ACBK01000034



Bryantella
formatexigens

ACCL02000018



Buchnera
aphidicola

NR_074609



Bulleidia
extructa

ADFR01000011



Burkholderia
ambifaria

AAUZ01000009



Burkholderia
cenocepacia

AAHI01000060



Burkholderia
cepacia

NR_041719



Burkholderia
mallei

CP000547



Burkholderia
multivorans

NC_010086



Burkholderia
oklahomensis

DQ108388



Burkholderia
pseudomallei

CP001408



Burkholderia
rhizoxinica

HQ005410



Burkholderia sp. 383
CP000151



Burkholderia
xenovorans

U86373



Burkholderiales bacterium 1_1_47
ADCQ01000066



Butyricicoccus
pullicaecorum

HH793440



Butyricimonas
virosa

AB443949



Butyrivibrio
crossotus

ABWN01000012



Butyrivibrio
fibrisolvens

U41172



Caldimonas
manganoxidans

NR_040787



Caminicella
sporogenes

NR_025485



Campylobacter
coli

AAFL01000004



Campylobacter
concisus

CP000792



Campylobacter
curvus

NC_009715


Campylobacter fetus
ACLG01001177



Campylobacter
gracilis

ACYG01000026



Campylobacter
hominis

NC_009714



Campylobacter
jejuni

AL139074



Campylobacter
lari

CP000932



Campylobacter
rectus

ACFU01000050



Campylobacter
showae

ACVQ01000030



Campylobacter sp. FOBRC14
HQ616379



Campylobacter sp. FOBRC15
HQ616380



Campylobacter sp. oral clone BB120
AY005038



Campylobacter
sputorum

NR_044839



Campylobacter
upsaliensis

AEPU01000040



Candidatus
Arthromitus sp. SFB_mouse_Yit
NR_074460



Candidatus
Sulcia
muelleri

CP002163



Capnocytophaga
canimorsus

CP002113



Capnocytophaga genomosp. C1
AY278613



Capnocytophaga
gingivalis

ACLQ01000011



Capnocytophaga
granulosa

X97248



Capnocytophaga
ochracea

AEOH01000054



Capnocytophaga sp. GEJ8
GU561335



Capnocytophaga sp. oral clone AH015
AY005074



Capnocytophaga sp. oral clone ASCH05
AY923149



Capnocytophaga sp. oral clone ID062
AY349368



Capnocytophaga sp. oral strain A47ROY
AY005077



Capnocytophaga sp. oral strain S3
AY005073



Capnocytophaga sp. oral taxon 338
AEXX01000050



Capnocytophaga sp. S1b
U42009



Capnocytophaga
sputigena

ABZV01000054



Cardiobacterium
hominis

ACKY01000036



Cardiobacterium
valvarum

NR_028847



Carnobacterium
divergens

NR_044706



Carnobacterium
maltaromaticum

NC_019425



Catabacter
hongkongensis

AB671763



Catenibacterium
mitsuokai

AB030224



Catonella genomosp. P1 oral clone MB5_P12
DQ003629



Catonella
morbi

ACIL02000016



Catonella sp. oral clone FL037
AY349369



Cedecea
davisae

AF493976



Cellulosimicrobium
funkei

AY501364



Cetobacterium
somerae

AJ438155



Chlamydia
muridarum

AE002160



Chlamydia
psittaci

NR_036864



Chlamydia
trachomatis

U68443



Chlamydiales bacterium NS11
JN606074



Chlamydiales bacterium NS13
JN606075



Chlamydiales bacterium NS16
JN606076



Chlamydophila
pecorum

D88317



Chlamydophila
pneumoniae

NC_002179



Chlamydophila
psittaci

D85712



Chloroflexi genomosp. P1
AY331414



Christensenella
minuta

AB490809



Chromobacterium
violaceum

NC_005085



Chryseobacterium
anthropi

AM982793



Chryseobacterium
gleum

ACKQ02000003



Chryseobacterium
hominis

NR_042517



Citrobacter
amalonaticus

FR870441



Citrobacter
braakii

NR_028687



Citrobacter
farmeri

AF025371



Citrobacter
freundii

NR_028894



Citrobacter
gillenii

AF025367



Citrobacter
koseri

NC_009792



Citrobacter
murliniae

AF025369



Citrobacter
rodentium

NR_074903



Citrobacter
sedlakii

AF025364



Citrobacter sp. 30_2
ACDJ01000053



Citrobacter sp. KMSI_3
GQ468398



Citrobacter
werkmanii

AF025373



Citrobacter
youngae

ABWL02000011



Cloacibacillus
evryensis

GQ258966



Clostridiaceae bacterium END_2
EF451053



Clostridiaceae bacterium JC13
JF824807



Clostridiales bacterium 1_7_47FAA
ABQR01000074



Clostridiales bacterium 9400853
HM587320



Clostridiales bacterium 9403326
HM587324



Clostridiales bacterium oral clone P4PA_66 P1
AY207065



Clostridiales bacterium oral taxon 093
GQ422712



Clostridiales bacterium oral taxon F32
HM099644



Clostridiales bacterium ph2
JN837487



Clostridiales bacterium SY8519
AB477431



Clostridiales genomosp. BVAB3
CP001850



Clostridiales sp. SM4_1
FP929060



Clostridiales sp. SS3_4
AY305316



Clostridiales sp. SSC_2
FP929061



Clostridium
acetobutylicum

NR_074511



Clostridium
aerotolerans

X76163



Clostridium
aldenense

NR_043680



Clostridium
aldrichii

NR_026099



Clostridium
algidicarnis

NR_041746



Clostridium
algidixylanolyticum

NR_028726



Clostridium
aminovalericum

NR_029245



Clostridium
amygdalinum

AY353957



Clostridium
argentinense

NR_029232



Clostridium
asparagiforme

ACCJ01000522



Clostridium
baratii

NR_029229



Clostridium
bartlettii

ABEZ02000012



Clostridium
beijerinckii

NR_074434



Clostridium
bifermentans

X73437



Clostridium
bolteae

ABCC02000039



Clostridium
botulinum

NC_010723



Clostridium
butyricum

ABDT01000017



Clostridium
cadaveris

AB542932



Clostridium
carboxidivorans

FR733710



Clostridium
carnis

NR_044716



Clostridium
celatum

X77844



Clostridium
celerecrescens

JQ246092



Clostridium
cellulosi

NR_044624



Clostridium
chauvoei

EU106372



Clostridium
citroniae

ADLJ01000059



Clostridium
clariflavum

NR_041235



Clostridium
clostridiiformes

M59089



Clostridium
clostridioforme

NR_044715



Clostridium
coccoides

EF025906



Clostridium
cochlearium

NR_044717



Clostridium
cocleatum

NR_026495



Clostridium
colicanis

FJ957863



Clostridium
colinum

NR_026151


Clostridium difficile
NC_013315



Clostridium
disporicum

NR_026491



Clostridium
estertheticum

NR_042153



Clostridium
fallax

NR_044714



Clostridium
favososporum

X76749



Clostridium
felsineum

AF270502



Clostridium
frigidicarnis

NR_024919



Clostridium
gasigenes

NR_024945



Clostridium
ghonii

AB542933



Clostridium
glycolicum

FJ384385



Clostridium
glycyrrhizinilyticum

AB233029



Clostridium
haemolyticum

NR_024749



Clostridium
hathewayi

AY552788



Clostridium
hiranonis

AB023970



Clostridium
histolyticum

HF558362



Clostridium
hylemonae

AB023973



Clostridium
indolis

AF028351



Clostridium
innocuum

M23732



Clostridium
irregulare

NR_029249



Clostridium
isatidis

NR_026347



Clostridium
kluyveri

NR_074165



Clostridium
lactatifermentans

NR_025651



Clostridium
lavalense

EF564277



Clostridium
leptum

AJ305238



Clostridium
limosum

FR870444



Clostridium
magnum

X77835



Clostridium
malenominatum

FR749893



Clostridium
mayombei

FR733682



Clostridium
methylpentosum

ACEC01000059



Clostridium
nexile

X73443



Clostridium
novyi

NR_074343



Clostridium
orbiscindens

Y18187



Clostridium
oroticum

FR749922



Clostridium
paraputrificum

AB536771



Clostridium
perfringens

ABDW01000023



Clostridium
phytofermentans

NR_074652



Clostridium
piliforme

D14639



Clostridium
putrefaciens

NR_024995



Clostridium
quinii

NR_026149



Clostridium
ramosum

M23731



Clostridium
rectum

NR_029271



Clostridium
saccharogumia

DQ100445



Clostridium
saccharolyticum

CP002109



Clostridium
sardiniense

NR_041006



Clostridium
sartagoforme

NR_026490



Clostridium
scindens

AF262238



Clostridium
septicum

NR_026020



Clostridium
sordellii

AB448946



Clostridium sp. 7_2_43FAA
ACDK01000101



Clostridium sp. D5
ADBG01000142



Clostridium sp. HGF2
AENW01000022



Clostridium sp. HPB_46
AY862516



Clostridium sp. JC122
CAEV01000127



Clostridium sp. L2_50
AAYW02000018



Clostridium sp. LMG 16094
X95274



Clostridium sp. M62_1
ACFX02000046



Clostridium sp. MLG055
AF304435



Clostridium sp. MT4 E
FJ159523



Clostridium sp. NMBHI_1
JN093130



Clostridium sp. NML 04A032
EU815224



Clostridium sp. SS2_1
ABGC03000041



Clostridium sp. SY8519
AP012212



Clostridium sp. TM_40
AB249652



Clostridium sp. YIT 12069
AB491207



Clostridium sp. YIT 12070
AB491208



Clostridium
sphenoides

X73449



Clostridium
spiroforme

X73441



Clostridium
sporogenes

ABKW02000003



Clostridium
sporosphaeroides

NR_044835



Clostridium
stercorarium

NR_025100



Clostridium
sticklandii

L04167



Clostridium
straminisolvens

NR_024829



Clostridium
subterminale

NR_041795



Clostridium
sulfidigenes

NR_044161



Clostridium
symbiosum

ADLQ01000114



Clostridium
tertium

Y18174



Clostridium
tetani

NC_004557



Clostridium
thermocellum

NR_074629



Clostridium
tyrobutyricum

NR_044718



Clostridium
viride

NR_026204



Clostridium
xylanolyticum

NR_037068



Collinsella
aerofaciens

AAVN02000007



Collinsella
intestinalis

ABXH02000037



Collinsella
stercoris

ABXJ01000150



Collinsella
tanakaei

AB490807



Comamonadaceae bacterium NML000135
JN585335



Comamonadaceae bacterium NML790751
JN585331



Comamonadaceae bacterium NML910035
JN585332



Comamonadaceae bacterium NML910036
JN585333



Comamonadaceae bacterium oral taxon F47
HM099651



Comamonas sp. NSP5
AB076850



Conchiformibius
kuhniae

NR_041821



Coprobacillus
cateniformis

AB030218



Coprobacillus sp. 29_1
ADKX01000057



Coprobacillus sp. D7
ACDT01000199



Coprococcus
catus

EU266552



Coprococcus
comes

ABVR01000038



Coprococcus
eutactus

EF031543



Coprococcus sp. ART55_1
AY350746



Coriobacteriaceae bacterium BV3Acl
JN809768



Coriobacteriaceae bacterium JC110
CAEM01000062



Coriobacteriaceae bacterium phI
JN837493



Corynebacterium
accolens

ACGD01000048



Corynebacterium
ammoniagenes

ADNS01000011



Corynebacterium
amycolatum

ABZU01000033



Corynebacterium
appendicis

NR_028951



Corynebacterium
argentoratense

EF463055



Corynebacterium
atypicum

NR_025540



Corynebacterium
aurimucosum

ACLH01000041



Corynebacterium
bovis

AF537590



Corynebacterium
canis

GQ871934



Corynebacterium
casei

NR_025101



Corynebacterium
confusum

Y15886



Corynebacterium
coyleae

X96497


Corynebacterium diphtheriae
NC_002935



Corynebacterium
durum

Z97069



Corynebacterium
efficiens

ACLI01000121



Corynebacterium
falsenii

Y13024



Corynebacterium
flavescens

NR_037040



Corynebacterium
genitalium

ACLJ01000031



Corynebacterium
glaucum

NR_028971



Corynebacterium
glucuronolyticum

ABYP01000081



Corynebacterium
glutamicum

BA000036



Corynebacterium
hansenii

AM946639



Corynebacterium
imitans

AF537597



Corynebacterium
jeikeium

ACYW01000001



Corynebacterium
kroppenstedtii

NR_026380



Corynebacterium
lipophiloflavum

ACHJ01000075



Corynebacterium
macginleyi

AB359393



Corynebacterium
mastitidis

AB359395



Corynebacterium
matruchotii

ACSH02000003



Corynebacterium
minutissimum

X82064



Corynebacterium
mucifaciens

NR_026396



Corynebacterium
propinquum

NR_037038



Corynebacterium
pseudodiphtheriticum

X84258



Corynebacterium
pseudogenitalium

ABYQ01000237



Corynebacterium
pseudotuberculosis

NR_037070



Corynebacterium
pyruviciproducens

FJ185225



Corynebacterium
renale

NR_037069



Corynebacterium
resistens

ADGN01000058



Corynebacterium
riegelii

EU848548



Corynebacterium
simulans

AF537604



Corynebacterium
singulare

NR_026394



Corynebacterium sp. 1 ex sheep
Y13427



Corynebacterium sp. L_2012475
HE575405



Corynebacterium sp. NML 93_0481
GU238409



Corynebacterium sp. NML 97_0186
GU238411



Corynebacterium sp. NML 99_0018
GU238413



Corynebacterium
striatum

ACGE01000001



Corynebacterium
sundsvallense

Y09655



Corynebacterium
tuberculostearicum

ACVP01000009



Corynebacterium
tuscaniae

AY677186



Corynebacterium
ulcerans

NR_074467



Corynebacterium
urealyticum

X81913



Corynebacterium
ureicelerivorans

AM397636



Corynebacterium
variabile

NR_025314



Corynebacterium
xerosis

FN179330



Coxiella
burnetii

CP000890



Cronobacter
malonaticus

GU122174



Cronobacter
sakazakii

NC_009778



Cronobacter
turicensis

FN543093



Cryptobacterium
curtum

GQ422741



Cupriavidus
metallidurans

GU230889



Cytophaga
xylanolytica

FR733683



Deferribacteres sp. oral clone JV001
AY349370



Deferribacteres sp. oral clone JV006
AY349371



Deferribacteres sp. oral clone JV023
AY349372



Deinococcus
radiodurans

AE000513



Deinococcus sp. R_43890
FR682752



Delftia
acidovorans

CP000884



Dermabacter
hominis

FJ263375



Dermacoccus sp. Ellin185
AEIQ01000090



Desmospora
activa

AM940019



Desmospora sp. 8437
AFHT01000143



Desulfitobacterium
frappieri

AJ276701



Desulfitobacterium
hafniense

NR_074996



Desulfobulbus sp. oral clone CH031
AY005036



Desulfotomaculum
nigrificans

NR_044832



Desulfovibrio
desulfuricans

DQ092636



Desulfovibrio
fairfieldensis

U42221



Desulfovibrio
piger

AF192152



Desulfovibrio sp. 3_1_syn3
ADDR01000239



Desulfovibrio
vulgaris

NR_074897



Dialister
invisus

ACIM02000001



Dialister
micraerophilus

AFBB01000028



Dialister
microaerophilus

AENT01000008



Dialister
pneumosintes

HM596297



Dialister
propionicifaciens

NR_043231



Dialister sp. oral taxon 502
GQ422739



Dialister
succinatiphilus

AB370249



Dietzia
natronolimnaea

GQ870426



Dietzia sp. BBDP51
DQ337512



Dietzia sp. CA149
GQ870422



Dietzia
timorensis

GQ870424



Dorea
formicigenerans

AAXA02000006



Dorea
longicatena

AJ132842



Dysgonomonas
gadei

ADLV01000001



Dysgonomonas
mossii

ADLW01000023



Edwardsiella
tarda

CP002154



Eggerthella
lenta

AF292375



Eggerthella
sinensis

AY321958



Eggerthella sp. 1_3_56FAA
ACWN01000099



Eggerthella sp. HGA1
AEXR01000021



Eggerthella sp. YY7918
AP012211



Ehrlichia
chaffeensis

AAIF01000035



Eikenella
corrodens

ACEA01000028



Enhydrobacter
aerosaccus

ACYI01000081



Enterobacter
aerogenes

AJ251468



Enterobacter
asburiae

NR_024640



Enterobacter
cancerogenus

Z96078



Enterobacter
cloacae

FP929040



Enterobacter
cowanii

NR_025566



Enterobacter
hormaechei

AFHR01000079



Enterobacter sp. 247BMC
HQ122932



Enterobacter sp. 638
NR_074777



Enterobacter sp. JC163
JN657217



Enterobacter sp. SCSS
HM007811



Enterobacter sp. TSE38
HM156134



Enterobacteriaceae bacterium 9_2_54FAA
ADCU01000033



Enterobacteriaceae bacterium CF01Ent_1
AJ489826



Enterobacteriaceae bacterium Smarlab 3302238
AY538694



Enterococcus
avium

AF133535



Enterococcus
caccae

AY943820



Enterococcus
casseliflavus

AEWT01000047



Enterococcus
durans

AJ276354



Enterococcus
faecalis

AE016830



Enterococcus
faecium

AM157434



Enterococcus
gallinarum

AB269767


Enterococcus gilvus
AY033814



Enterococcus
hawaiiensis

AY321377



Enterococcus
hirae

AF061011



Enterococcus
italicus

AEPV01000109



Enterococcus
mundtii

NR_024906



Enterococcus
raffinosus

FN600541



Enterococcus sp. BV2CASA2
JN809766



Enterococcus sp. CCRI_16620
GU457263



Enterococcus sp. F95
FJ463817



Enterococcus sp. RfL6
AJ133478



Enterococcus
thailandicus

AY321376



Eremococcus
coleocola

AENN01000008



Erysipelothrix
inopinata

NR_025594



Erysipelothrix
rhusiopathiae

ACLK01000021



Erysipelothrix
tonsillarum

NR_040871



Erysipelotrichaceae bacterium 3_1_53
ACTJ01000113



Erysipelotrichaceae bacterium 5_2_54FAA
ACZW01000054



Escherichia
albertii

ABKX01000012



Escherichia
coli

NC_008563



Escherichia
fergusonii

CU928158



Escherichia
hermannii

HQ407266



Escherichia sp. 1_1_43
ACID01000033



Escherichia sp. 4_1_40B
ACDM02000056



Escherichia sp. B4
EU722735



Escherichia
vulneris

NR_041927



Ethanoligenens
harbinense

AY675965



Eubacteriaceae bacterium P4P_50 P4
AY207060



Eubacterium
barkeri

NR_044661



Eubacterium
biforme

ABYT01000002



Eubacterium
brachy

U13038



Eubacterium
budayi

NR_024682



Eubacterium
callanderi

NR_026330



Eubacterium
cellulosolvens

AY178842



Eubacterium
contortum

FR749946



Eubacterium
coprostanoligenes

HM037995



Eubacterium
cylindroides

FP929041



Eubacterium
desmolans

NR_044644



Eubacterium
dolichum

L34682



Eubacterium
eligens

CP001104



Eubacterium
fissicatena

FR749935



Eubacterium
hadrum

FR749933



Eubacterium
hallii

L34621



Eubacterium
infirmum

U13039



Eubacterium
limosum

CP002273



Eubacterium
moniliforme

HF558373



Eubacterium
multiforme

NR_024683



Eubacterium
nitritogenes

NR_024684



Eubacterium
nodatum

U13041



Eubacterium
ramulus

AJ011522



Eubacterium
rectale

FP929042



Eubacterium
ruminantium

NR_024661



Eubacterium
saburreum

AB525414



Eubacterium
saphenum

NR_026031



Eubacterium
siraeum

ABCA03000054



Eubacterium sp. 3_1_31
ACTL01000045



Eubacterium sp. AS15b
HQ616364



Eubacterium sp. OBRC9
HQ616354



Eubacterium sp. oral clone GI038
AY349374



Eubacterium sp. oral clone IR009
AY349376



Eubacterium sp. oral clone JH012
AY349373



Eubacterium sp. oral clone JI012
AY349379



Eubacterium sp. oral clone JN088
AY349377



Eubacterium sp. oral clone JS001
AY349378



Eubacterium sp. oral clone OH3A
AY947497



Eubacterium sp. WAL 14571
FJ687606



Eubacterium
tenue

M59118



Eubacterium
tortuosum

NR_044648



Eubacterium
ventriosum

L34421



Eubacterium
xylanophilum

L34628



Eubacterium
yurii

AEES01000073



Ewingella
americana

JN175329



Exiguobacterium
acetylicum

FJ970034



Facklamia
hominis

Y10772



Faecalibacterium
prausnitzii

ACOP02000011



Filifactor
alocis

CP002390



Filifactor
villosus

NR_041928



Finegoldia
magna

ACHM02000001



Flavobacteriaceae genomosp. C1
AY278614



Flavobacterium sp. NF2_1
FJ195988



Flavonifractor
plautii

AY724678



Flexispira
rappini

AY126479



Flexistipes
sinusarabici

NR_074881



Francisella
novicida

ABSS01000002



Francisella
philomiragia

AY928394



Francisella
tularensis

ABAZ01000082



Fulvimonas sp. NML 060897
EF589680



Fusobacterium
canifelinum

AY162222



Fusobacterium genomosp. C1
AY278616



Fusobacterium genomosp. C2
AY278617



Fusobacterium
gonidiaformans

ACET01000043



Fusobacterium
mortiferum

ACDB02000034



Fusobacterium
naviforme

HQ223106



Fusobacterium
necrogenes

X55408



Fusobacterium
necrophorum

AM905356



Fusobacterium
nucleatum

ADVK01000034



Fusobacterium
periodonticum

ACJY01000002



Fusobacterium
russii

NR_044687



Fusobacterium sp. 1_1_41FAA
ADGG01000053



Fusobacterium sp. 11_3_2
ACUO01000052



Fusobacterium sp. 12_1B
AGWJ01000070



Fusobacterium sp. 2_1_31
ACDC02000018



Fusobacterium sp. 3_1_27
ADGF01000045



Fusobacterium sp. 3_1_33
ACQE01000178



Fusobacterium sp. 3_1_36A2
ACPU01000044



Fusobacterium sp. 3_1_5R
ACDD01000078



Fusobacterium sp. AC18
HQ616357



Fusobacterium sp. ACB2
HQ616358



Fusobacterium sp. AS2
HQ616361



Fusobacterium sp. CM1
HQ616371



Fusobacterium sp. CM21
HQ616375



Fusobacterium sp. CM22
HQ616376



Fusobacterium sp. D12
ACDG02000036



Fusobacterium sp. oral clone ASCF06
AY923141



Fusobacterium sp. oral clone ASCF11
AY953256



Fusobacterium
ulcerans

ACDH01000090



Fusobacterium
varium

ACIE01000009



Gardnerella
vaginalis

CP001849


Gemella haemolysans
ACDZ02000012



Gemella
morbillorum

NR_025904



Gemella
morbillorum

ACRX01000010



Gemella
sanguinis

ACRY01000057



Gemella sp. oral clone ASCE02
AY923133



Gemella sp. oral clone ASCF04
AY923139



Gemella sp. oral clone ASCF12
AY923143



Gemella sp. WAL 1945J
EU427463



Gemmiger
formicilis

GU562446



Geobacillus
kaustophilus

NR_074989



Geobacillus sp. E263
DQ647387



Geobacillus sp. WCH70
CP001638



Geobacillus
stearothermophilus

NR_040794



Geobacillus
thermocatenulatus

NR_043020



Geobacillus
thermodenitrificans

NR_074976



Geobacillus
thermoglucosidasius

NR_043022



Geobacillus
thermoleovorans

NR_074931



Geobacter
bemidjiensis

CP001124



Gloeobacter
violaceus

NR_074282



Gluconacetobacter
azotocaptans

NR_028767



Gluconacetobacter
diazotrophicus

NR_074292



Gluconacetobacter
entanii

NR_028909



Gluconacetobacter
europaeus

NR_026513



Gluconacetobacter
hansenii

NR_026133



Gluconacetobacter
johannae

NR_024959



Gluconacetobacter
oboediens

NR_041295



Gluconacetobacter
xylinus

NR_074338



Gordonia
bronchialis

NR_027594



Gordonia
polyisoprenivorans

DQ385609



Gordonia sp. KTR9
DQ068383



Gordonia
sputi

FJ536304



Gordonia
terrae

GQ848239



Gordonibacter
pamelaeae

AM886059



Gordonibacter
pamelaeae

FP929047



Gracilibacter
thermotolerans

NR_043559



Gramella
forsetii

NR_074707



Granulicatella
adiacens

ACKZ01000002



Granulicatella
elegans

AB252689



Granulicatella
paradiacens

AY879298



Granulicatella sp. M658_99_3
AJ271861



Granulicatella sp. oral clone ASC02
AY923126



Granulicatella sp. oral clone ASCA05
DQ341469



Granulicatella sp. oral clone ASCB09
AY953251



Granulicatella sp. oral clone ASCG05
AY923146



Grimontia
hollisae

ADAQ01000013



Haematobacter sp. BC14248
GU396991



Haemophilus
aegyptius

AFBC01000053



Haemophilus
ducreyi

AE017143



Haemophilus genomosp. P2 oral clone
DQ003621


MB3_C24




Haemophilus genomosp. P3 oral clone
DQ003635


MB3_C38




Haemophilus
haemolyticus

JN175335



Haemophilus
influenzae

AADP01000001



Haemophilus
parahaemolyticus

GU561425



Haemophilus
parainfluenzae

AEWU01000024



Haemophilus
paraphrophaemolyticus

M75076



Haemophilus
parasuis

GU226366



Haemophilus
somnus

NC_008309



Haemophilus sp. 70334
HQ680854



Haemophilus sp. HK445
FJ685624



Haemophilus sp. oral clone ASCA07
AY923117



Haemophilus sp. oral clone ASCG06
AY923147



Haemophilus sp. oral clone BJ021
AY005034



Haemophilus sp. oral clone BJ095
AY005033



Haemophilus sp. oral clone JM053
AY349380



Haemophilus sp. oral taxon 851
AGRK01000004



Haemophilus
sputorum

AFNK01000005



Hafnia
alvei

DQ412565



Halomonas
elongata

NR_074782



Halomonas
johnsoniae

FR775979



Halorubrum
lipolyticum

AB477978



Helicobacter
bilis

ACDN01000023



Helicobacter
canadensis

ABQS01000108



Helicobacter
cinaedi

ABQT01000054



Helicobacter
pullorum

ABQU01000097



Helicobacter
pylori

CP000012



Helicobacter sp. None
U44756



Helicobacter
winghamensis

ACDO01000013



Heliobacterium
modesticaldum

NR_074517



Herbaspirillum
seropedicae

CP002039



Herbaspirillum sp. JC206
JN657219



Histophilus
somni

AF549387



Holdemania
filiformis

Y11466



Hydrogenoanaerobacterium
saccharovorans

NR_044425



Hyperthermus
butylicus

CP000493



Hyphomicrobium
sulfonivorans

AY468372



Hyphomonas
neptunium

NR_074092



Ignatzschineria
indica

HQ823562



Ignatzschineria sp. NML 95_0260
HQ823559



Ignicoccus
islandicus

X99562



Inquilinus
limosus

NR_029046



Janibacter
limosus

NR_026362



Janibacter
melonis

EF063716



Janthinobacterium sp. SY12
EF455530



Johnsonella
ignava

X87152



Jonquetella
anthropi

ACOO02000004



Kerstersia
gyiorum

NR_025669



Kingella
denitrificans

AEWV01000047



Kingella genomosp. P1 oral cone MB2_C20
DQ003616



Kingella
kingae

AFHS01000073



Kingella
oralis

ACJW02000005



Kingella sp. oral clone ID059
AY349381



Klebsiella
oxytoca

AY292871



Klebsiella
pneumoniae

CP000647



Klebsiella sp. AS10
HQ616362



Klebsiella sp. Co9935
DQ068764



Klebsiella sp. enrichment culture clone
HM195210


SRC_DSD25




Klebsiella sp. OBRC7
HQ616353



Klebsiella sp. SP_BA
FJ999767



Klebsiella sp. SRC_DSD1
GU797254


Klebsiella sp. SRC_DSD11
GU797263



Klebsiella sp. SRC_DSD12
GU797264



Klebsiella sp. SRC_DSD15
GU797267



Klebsiella sp. SRC_DSD2
GU797253



Klebsiella sp. SRC_DSD6
GU797258



Klebsiella
variicola

CP001891



Kluyvera
ascorbata

NR_028677



Kluyvera
cryocrescens

NR_028803



Kocuria
marina

GQ260086



Kocuria
palustris

EU333884



Kocuria
rhizophila

AY030315



Kocuria
rosea

X87756



Kocuria
varians

AF542074



Lachnobacterium
bovis

GU324407



Lachnospira
multipara

FR733699



Lachnospira
pectinoschiza

L14675



Lachnospiraceae bacterium 1_1_57FAA
ACTM01000065



Lachnospiraceae bacterium 1_4_56FAA
ACTN01000028



Lachnospiraceae bacterium 2_1_46FAA
ADLB01000035



Lachnospiraceae bacterium 2_1_58FAA
ACTO01000052



Lachnospiraceae bacterium 3_1_57FAA_CT1
ACTP01000124



Lachnospiraceae bacterium 4_1_37FAA
ADCR01000030



Lachnospiraceae bacterium 5_1_57FAA
ACTR01000020



Lachnospiraceae bacterium 5_1_63FAA
ACTS01000081



Lachnospiraceae bacterium 6_1_63FAA
ACTV01000014



Lachnospiraceae bacterium 8_1_57FAA
ACWQ01000079



Lachnospiraceae bacterium 9_1_43BFAA
ACTX01000023



Lachnospiraceae bacterium A4
DQ789118



Lachnospiraceae bacterium DJF VP30
EU728771



Lachnospiraceae bacterium ICM62
HQ616401



Lachnospiraceae bacterium MSX33
HQ616384



Lachnospiraceae bacterium oral taxon 107
ADDS01000069



Lachnospiraceae bacterium oral taxon F15
HM099641



Lachnospiraceae genomosp. C1
AY278618



Lactobacillus
acidipiscis

NR_024718



Lactobacillus
acidophilus

CP000033



Lactobacillus
alimentarius

NR_044701



Lactobacillus
amylolyticus

ADNY01000006



Lactobacillus
amylovorus

CP002338



Lactobacillus
antri

ACLL01000037



Lactobacillus
brevis

EU194349



Lactobacillus
buchneri

ACGH01000101



Lactobacillus
casei

CP000423



Lactobacillus
catenaformis

M23729



Lactobacillus
coleohominis

ACOH01000030



Lactobacillus
coryniformis

NR_044705



Lactobacillus
crispatus

ACOG01000151



Lactobacillus
curvatus

NR_042437



Lactobacillus
delbrueckii

CP002341



Lactobacillus
dextrinicus

NR_036861



Lactobacillus
farciminis

NR_044707



Lactobacillus
fermentum

CP002033



Lactobacillus
gasseri

ACOZ01000018



Lactobacillus
gastricus

AICN01000060



Lactobacillus genomosp. C1
AY278619



Lactobacillus genomosp. C2
AY278620



Lactobacillus
helveticus

ACLM01000202



Lactobacillus
hilgardii

ACGP01000200



Lactobacillus
hominis

FR681902



Lactobacillus
iners

AEKJ01000002



Lactobacillus
jensenii

ACQD01000066



Lactobacillus
johnsonii

AE017198



Lactobacillus
kalixensis

NR_029083



Lactobacillus
kefiranofaciens

NR_042440



Lactobacillus
kefiri

NR_042230



Lactobacillus
kimchii

NR_025045



Lactobacillus
leichmannii

JX986966



Lactobacillus
mucosae

FR693800



Lactobacillus
murinus

NR_042231



Lactobacillus
nodensis

NR_041629



Lactobacillus
oeni

NR_043095



Lactobacillus
oris

AEKL01000077



Lactobacillus
parabrevis

NR_042456



Lactobacillus
parabuchneri

NR_041294



Lactobacillus
paracasei

ABQV01000067



Lactobacillus
parakefiri

NR_029039



Lactobacillus
pentosus

JN813103



Lactobacillus
perolens

NR_029360



Lactobacillus
plantarum

ACGZ02000033



Lactobacillus
pontis

HM218420



Lactobacillus
reuteri

ACGW02000012



Lactobacillus
rhamnosus

ABWJ01000068



Lactobacillus
rogosae

GU269544



Lactobacillus
ruminis

ACGS02000043



Lactobacillus
sakei

DQ989236



Lactobacillus
salivarius

AEBA01000145



Lactobacillus
saniviri

AB602569



Lactobacillus
senioris

AB602570



Lactobacillus sp. 66c
FR681900



Lactobacillus sp. BT6
HQ616370



Lactobacillus sp. KLDS 1.0701
EU600905



Lactobacillus sp. KLDS 1.0702
EU600906



Lactobacillus sp. KLDS 1.0703
EU600907



Lactobacillus sp. KLDS 1.0704
EU600908



Lactobacillus sp. KLDS 1.0705
EU600909



Lactobacillus sp. KLDS 1.0707
EU600911



Lactobacillus sp. KLDS 1.0709
EU600913



Lactobacillus sp. KLDS 1.0711
EU600915



Lactobacillus sp. KLDS 1.0712
EU600916



Lactobacillus sp. KLDS 1.0713
EU600917



Lactobacillus sp. KLDS 1.0716
EU600921



Lactobacillus sp. KLDS 1.0718
EU600922



Lactobacillus sp. KLDS 1.0719
EU600923



Lactobacillus sp. oral clone HT002
AY349382



Lactobacillus sp. oral clone HT070
AY349383



Lactobacillus sp. oral taxon 052
GQ422710



Lactobacillus
tucceti

NR_042194



Lactobacillus
ultunensis

ACGU01000081


Lactobacillus vaginalis
ACGV01000168



Lactobacillus
vini

NR_042196



Lactobacillus
vitulinus

NR_041305



Lactobacillus
zeae

NR_037122



Lactococcus
garvieae

AF061005



Lactococcus
lactis

CP002365



Lactococcus
raffinolactis

NR_044359



Lactonifactor
longoviformis

DQ100449



Laribacter
hongkongensis

CP001154



Lautropia
mirabilis

AEQP01000026



Lautropia sp. oral clone AP009
AY005030



Legionella
hackeliae

M36028



Legionella
longbeachae

M36029



Legionella
pneumophila

NC_002942



Legionella sp. D3923
JN380999



Legionella sp. D4088
JN381012



Legionella sp. H63
JF831047



Legionella sp. NML 93L054
GU062706



Legionella
steelei

HQ398202



Leminorella
grimontii

AJ233421



Leminorella
richardii

HF558368



Leptospira
borgpetersenii

NC_008508



Leptospira
broomii

NR_043200



Leptospira
interrogans

NC_005823



Leptospira
licerasiae

EF612284



Leptotrichia
buccalis

CP001685



Leptotrichia genomosp. C1
AY278621



Leptotrichia
goodfellowii

ADAD01000110



Leptotrichia
hofstadii

ACVB02000032



Leptotrichia
shahii

AY029806



Leptotrichia sp. neutropenicPatient
AF189244



Leptotrichia sp. oral clone GT018
AY349384



Leptotrichia sp. oral clone GT020
AY349385



Leptotrichia sp. oral clone HE012
AY349386



Leptotrichia sp. oral clone IK040
AY349387



Leptotrichia sp. oral clone P2PB_51 P1
AY207053



Leptotrichia sp. oral taxon 223
GU408547



Leuconostoc
carnosum

NR_040811



Leuconostoc
citreum

AM157444



Leuconostoc
gasicomitatum

FN822744



Leuconostoc
inhae

NR_025204



Leuconostoc
kimchii

NR_075014



Leuconostoc
lactis

NR_040823



Leuconostoc
mesenteroides

ACKV01000113



Leuconostoc
pseudomesenteroides

NR_040814



Listeria
grayi

ACCR02000003



Listeria
innocua

JF967625



Listeria
ivanovii

X56151



Listeria
monocytogenes

CP002003



Listeria
welshimeri

AM263198



Luteococcus
sanguinis

NR_025507



Lutispora
thermophila

NR_041236



Lysinibacillus
fusiformis

FN397522



Lysinibacillus
sphaericus

NR_074883



Macrococcus
caseolyticus

NR_074941



Mannheimia
haemolytica

ACZX01000102



Marvinbryantia
formatexigens

AJ505973



Massilia sp. CCUG 43427A
FR773700



Megamonas
funiformis

AB300988



Megamonas
hypermegale

AJ420107



Megasphaera
elsdenii

AY038996



Megasphaera genomosp. C1
AY278622



Megasphaera genomosp. type_1
ADGP01000010



Megasphaera
micronuciformis

AECS01000020



Megasphaera sp. BLPYG_07
HM990964



Megasphaera sp. UPII 199_6
AFIJ01000040



Metallosphaera
sedula

D26491



Methanobacterium
formicicum

NR_025028



Methanobrevibacter
acididurans

NR_028779



Methanobrevibacter
arboriphilus

NR_042783



Methanobrevibacter
curvatus

NR_044796



Methanobrevibacter
cuticularis

NR_044776



Methanobrevibacter
filiformis

NR_044801



Methanobrevibacter
gottschalkii

NR_044789



Methanobrevibacter
millerae

NR_042785



Methanobrevibacter
olleyae

NR_043024



Methanobrevibacter
oralis

HE654003



Methanobrevibacter
ruminantium

NR_042784



Methanobrevibacter
smithii

ABYV02000002



Methanobrevibacter
thaueri

NR_044787



Methanobrevibacter
woesei

NR_044788



Methanobrevibacter
wolinii

NR_044790



Methanosphaera
stadtmanae

AY196684



Methylobacterium
extorquens

NC_010172



Methylobacterium
podarium

AY468363



Methylobacterium
radiotolerans

GU294320



Methylobacterium sp. 1sub
AY468371



Methylobacterium sp. MM4
AY468370



Methylocella
silvestris

NR_074237



Methylophilus sp. ECd5
AY436794



Microbacterium
chocolatum

NR_037045



Microbacterium
flavescens

EU714363



Microbacterium
gubbeenense

NR_025098



Microbacterium
lacticum

EU714351



Microbacterium
oleivorans

EU714381



Microbacterium
oxydans

EU714348



Microbacterium
paraoxydans

AJ491806



Microbacterium
phyllosphaerae

EU714359



Microbacterium
schleiferi

NR_044936



Microbacterium sp. 768
EU714378



Microbacterium sp. oral strain C24KA
AF287752



Microbacterium
testaceum

EU714365



Micrococcus
antarcticus

NR_025285



Micrococcus
luteus

NR_075062



Micrococcus
lylae

NR_026200



Micrococcus sp. 185
EU714334



Microcystis
aeruginosa

NC_010296



Mitsuokella
jalaludinii

NR_028840



Mitsuokella
multacida

ABWK02000005



Mitsuokella sp. oral taxon 521
GU413658



Mitsuokella sp. oral taxon G68
GU432166



Mobiluncus
curtisii

AEPZ01000013



Mobiluncus
mulieris

ACKW01000035



Moellerella
wisconsensis

JN175344



Mogibacterium
diversum

NR_027191



Mogibacterium
neglectum

NR_027203



Mogibacterium
pumilum

NR_028608



Mogibacterium
timidum

Z36296



Mollicutes bacterium pACH93
AY297808



Moorella
thermoacetica

NR_075001



Moraxella
catarrhalis

CP002005



Moraxella
lincolnii

FR822735



Moraxella
osloensis

JN175341


Moraxella sp. 16285
JF682466



Moraxella sp. GM2
JF837191



Morganella
morganii

AJ301681



Morganella sp. JB_T16
AJ781005



Morococcus
cerebrosus

JN175352



Moryella
indoligenes

AF527773



Mycobacterium
abscessus

AGQU01000002



Mycobacterium
africanum

AF480605



Mycobacterium
alsiensis

AJ938169



Mycobacterium
avium

CP000479



Mycobacterium
chelonae

AB548610



Mycobacterium
colombiense

AM062764



Mycobacterium
elephantis

AF385898



Mycobacterium
gordonae

GU142930



Mycobacterium
intracellulare

GQ153276



Mycobacterium
kansasii

AF480601



Mycobacterium
lacus

NR_025175



Mycobacterium
leprae

FM211192



Mycobacterium
lepromatosis

EU203590



Mycobacterium
mageritense

FR798914



Mycobacterium
mantenii

FJ042897



Mycobacterium
marinum

NC_010612



Mycobacterium
microti

NR_025234



Mycobacterium
neoaurum

AF268445



Mycobacterium
parascrofulaceum

ADNV01000350



Mycobacterium
paraterrae

EU919229



Mycobacterium
phlei

GU142920



Mycobacterium
seoulense

DQ536403



Mycobacterium
smegmatis

CP000480



Mycobacterium sp. 1761
EU703150



Mycobacterium sp. 1776
EU703152



Mycobacterium sp. 1781
EU703147



Mycobacterium sp. 1791
EU703148



Mycobacterium sp. 1797
EU703149



Mycobacterium sp. AQIGA4
HM210417



Mycobacterium sp. B10_07.09.0206
HQ174245



Mycobacterium sp. GN_10546
FJ497243



Mycobacterium sp. GN_10827
FJ497247



Mycobacterium sp. GN_11124
FJ652846



Mycobacterium sp. GN_9188
FJ497240



Mycobacterium sp. GR_2007_210
FJ555538



Mycobacterium sp. HE5
AJ012738



Mycobacterium sp. NLA001000736
HM627011



Mycobacterium sp. W
DQ437715



Mycobacterium
tuberculosis

CP001658



Mycobacterium
ulcerans

AB548725



Mycobacterium
vulneris

EU834055



Mycoplasma
agalactiae

AF010477



Mycoplasma
amphoriforme

AY531656



Mycoplasma
arthritidis

NC_011025



Mycoplasma
bovoculi

NR_025987



Mycoplasma
faucium

NR_024983



Mycoplasma
fermentans

CP002458



Mycoplasma
flocculare

X62699



Mycoplasma
genitalium

L43967



Mycoplasma
hominis

AF443616



Mycoplasma
orale

AY796060



Mycoplasma
ovipneumoniae

NR_025989



Mycoplasma
penetrans

NC_004432



Mycoplasma
pneumoniae

NC_000912



Mycoplasma
putrefaciens

U26055



Mycoplasma
salivarium

M24661



Mycoplasmataceae genomosp. P1 oral clone
DQ003614


MB1_G23




Myroides
odoratimimus

NR_042354



Myroides sp. MY15
GU253339



Neisseria
bacilliformis

AFAY01000058



Neisseria
cinerea

ACDY01000037



Neisseria
elongata

ADBF01000003



Neisseria
flavescens

ACQV01000025



Neisseria genomosp. P2 oral clone MB5_P15
DQ003630



Neisseria
gonorrhoeae

CP002440



Neisseria
lactamica

ACEQ01000095



Neisseria
macacae

AFQE01000146



Neisseria
meningitidis

NC_003112



Neisseria
mucosa

ACDX01000110



Neisseria
pharyngis

AJ239281



Neisseria
polysaccharea

ADBE01000137



Neisseria
sicca

ACKO02000016



Neisseria sp. KEM232
GQ203291



Neisseria sp. oral clone AP132
AY005027



Neisseria sp. oral clone JC012
AY349388



Neisseria sp. oral strain B33KA
AY005028



Neisseria sp. oral taxon 014
ADEA01000039



Neisseria sp. SMC_A9199
FJ763637



Neisseria sp. TM10_1
DQ279352



Neisseria
subflava

ACEO01000067



Neorickettsia
risticii

CP001431



Neorickettsia
sennetsu

NC_007798



Nocardia
brasiliensis

AIHV01000038



Nocardia
cyriacigeorgica

HQ009486



Nocardia
farcinica

NC_006361



Nocardia
puris

NR_028994



Nocardia sp. 01_Je_025
GU574059



Nocardiopsis
dassonvillei

CP002041



Novosphingobium
aromaticivorans

AAAV03000008



Oceanobacillus
caeni

NR_041533



Oceanobacillus sp. Ndiop
CAER01000083



Ochrobactrum
anthropi

NC_009667



Ochrobactrum
intermedium

ACQA01000001



Ochrobactrum
pseudintermedium

DQ365921



Odoribacter
laneus

AB490805



Odoribacter
splanchnicus

CP002544



Okadaella
gastrococcus

HQ699465



Oligella
ureolytica

NR_041998



Oligella
urethralis

NR_041753



Olsenella genomosp. C1
AY278623



Olsenella
profusa

FN178466



Olsenella sp. F0004
EU592964



Olsenella sp. oral taxon 809
ACVE01000002



Olsenella
uli

CP002106



Opitutus
terrae

NR_074978



Oribacterium
sinus

ACKX01000142



Oribacterium sp. ACB1
HM120210



Oribacterium sp. ACB7
HM120211



Oribacterium sp. CM12
HQ616374



Oribacterium sp. ICM51
HQ616397



Oribacterium sp. OBRC12
HQ616355



Oribacterium sp. oral taxon 078
ACIQ02000009



Oribacterium sp. oral taxon 102
GQ422713


Oribacterium sp. oral taxon 108
AFIH01000001



Orientia
tsutsugamushi

AP008981



Ornithinibacillus
bavariensis

NR_044923



Ornithinibacillus sp. 7_10AIA
FN397526



Oscillibacter sp. G2
HM626173



Oscillibacter
valericigenes

NR_074793



Oscillospira
guilliermondii

AB040495



Oxalobacter
formigenes

ACDQ01000020



Paenibacillus
barcinonensis

NR_042272



Paenibacillus
barengoltzii

NR_042756



Paenibacillus
chibensis

NR_040885



Paenibacillus
cookii

NR_025372



Paenibacillus
durus

NR_037017



Paenibacillus
glucanolyticus

D78470



Paenibacillus
lactis

NR_025739



Paenibacillus
lautus

NR_040882



Paenibacillus
pabuli

NR_040853



Paenibacillus
polymyxa

NR_037006



Paenibacillus
popilliae

NR_040888



Paenibacillus sp. CIP 101062
HM212646



Paenibacillus sp. HGF5
AEXS01000095



Paenibacillus sp. HGF7
AFDH01000147



Paenibacillus sp. JC66
JF824808



Paenibacillus sp. oral taxon F45
HM099647



Paenibacillus sp. R_27413
HE586333



Paenibacillus sp. R_27422
HE586338



Paenibacillus
timonensis

NR_042844



Pantoea
agglomerans

AY335552



Pantoea
ananatis

CP001875



Pantoea
brenneri

EU216735



Pantoea
citrea

EF688008



Pantoea
conspicua

EU216737



Pantoea
septica

EU216734



Papillibacter
cinnamivorans

NR_025025



Parabacteroides
distasonis

CP000140



Parabacteroides
goldsteinii

AY974070



Parabacteroides
gordonii

AB470344



Parabacteroides
johnsonii

ABYH01000014



Parabacteroides
merdae

EU136685



Parabacteroides sp. D13
ACPW01000017



Parabacteroides sp. NS31_3
JN029805



Parachlamydia sp. UWE25
BX908798



Paracoccus
denitrificans

CP000490



Paracoccus
marcusii

NR_044922



Paraprevotella
clara

AFFY01000068



Paraprevotella
xylaniphila

AFBR01000011



Parascardovia
denticolens

ADEB01000020



Parasutterella
excrementihominis

AFBP01000029



Parasutterella
secunda

AB491209



Parvimonas
micra

AB729072



Parvimonas sp. oral taxon 110
AFII01000002



Pasteurella
bettyae

L06088



Pasteurella
dagmatis

ACZR01000003



Pasteurella
multocida

NC_002663



Pediococcus
acidilactici

ACXB01000026



Pediococcus
pentosaceus

NR_075052



Peptococcus
niger

NR_029221



Peptococcus sp. oral clone JM048
AY349389



Peptococcus sp. oral taxon 167
GQ422727



Peptoniphilus
asaccharolyticus

D14145



Peptoniphilus
duerdenii

EU526290



Peptoniphilus
harei

NR_026358



Peptoniphilus
indolicus

AY153431



Peptoniphilus
ivorii

Y07840



Peptoniphilus
lacrimalis

ADDO01000050



Peptoniphilus sp. gpac007
AM176517



Peptoniphilus sp. gpac018A
AM176519



Peptoniphilus sp. gpac077
AM176527



Peptoniphilus sp. gpac148
AM176535



Peptoniphilus sp. JC140
JF824803



Peptoniphilus sp. oral taxon 386
ADCS01000031



Peptoniphilus sp. oral taxon 836
AEAA01000090



Peptostreptococcaceae bacterium phl
JN837495



Peptostreptococcus
anaerobius

AY326462



Peptostreptococcus
micros

AM176538



Peptostreptococcus sp. 9succ1
X90471



Peptostreptococcus sp. oral clone AP24
AB175072



Peptostreptococcus sp. oral clone FJ023
AY349390



Peptostreptococcus sp. P4P_31 P3
AY207059



Peptostreptococcus
stomatis

ADGQ01000048



Phascolarctobacterium
faecium

NR_026111



Phascolarctobacterium sp. YIT 12068
AB490812



Phascolarctobacterium
succinatutens

AB490811



Phenylobacterium
zucineum

AY628697



Photorhabdus
asymbiotica

Z76752



Pigmentiphaga
daeguensis

JN585327



Planomicrobium
koreense

NR_025011



Plesiomonas
shigelloides

X60418



Porphyromonadaceae bacterium NML 060648
EF184292



Porphyromonas
asaccharolytica

AENO01000048



Porphyromonas
endodontalis

ACNN01000021



Porphyromonas
gingivalis

AE015924



Porphyromonas
levii

NR_025907



Porphyromonas
macacae

NR_025908



Porphyromonas
somerae

AB547667



Porphyromonas sp. oral clone BB134
AY005068



Porphyromonas sp. oral clone F016
AY005069



Porphyromonas sp. oral clone P2PB_52 P1
AY207054



Porphyromonas sp. oral clone P4GB_100 P2
AY207057



Porphyromonas sp. UQD 301
EU012301



Porphyromonas
uenonis

ACLR01000152



Prevotella
albensis

NR_025300



Prevotella
amnii

AB547670



Prevotella
bergensis

ACKS01000100



Prevotella
bivia

ADFO01000096



Prevotella
brevis

NR_041954



Prevotella
buccae

ACRB01000001



Prevotella
buccalis

JN867261



Prevotella
copri

ACBX02000014



Prevotella
corporis

L16465



Prevotella
dentalis

AB547678



Prevotella
denticola

CP002589



Prevotella
disiens

AEDO01000026



Prevotella genomosp. C1
AY278624



Prevotella genomosp. C2
AY278625


Prevotella genomosp. P7 oral clone MB2_P31
DQ003620



Prevotella genomosp. P8 oral clone MB3_P13
DQ003622



Prevotella genomosp. P9 oral clone MB7_G16
DQ003633



Prevotella
heparinolytica

GQ422742



Prevotella
histicola

JN867315



Prevotella
intermedia

AF414829



Prevotella
loescheii

JN867231



Prevotella
maculosa

AGEK01000035



Prevotella
marshii

AEEI01000070



Prevotella
melaninogenica

CP002122



Prevotella
micans

AGWK01000061



Prevotella
multiformis

AEWX01000054



Prevotella
multisaccharivorax

AFJE01000016



Prevotella
nanceiensis

JN867228



Prevotella
nigrescens

AFPX01000069



Prevotella
oralis

AEPE01000021



Prevotella
oris

ADDV01000091



Prevotella
oulorum

L16472



Prevotella
pallens

AFPY01000135



Prevotella
ruminicola

CP002006



Prevotella
salivae

AB108826



Prevotella sp. BI_42
AJ581354



Prevotella sp. CM38
HQ610181



Prevotella sp. ICM1
HQ616385



Prevotella sp. ICM55
HQ616399



Prevotella sp. JCM 6330
AB547699



Prevotella sp. oral clone AA020
AY005057



Prevotella sp. oral clone ASCG10
AY923148



Prevotella sp. oral clone ASCG12
DQ272511



Prevotella sp. oral clone AU069
AY005062



Prevotella sp. oral clone CY006
AY005063



Prevotella sp. oral clone DA058
AY005065



Prevotella sp. oral clone FL019
AY349392



Prevotella sp. oral clone FU048
AY349393



Prevotella sp. oral clone FW035
AY349394



Prevotella sp. oral clone GI030
AY349395



Prevotella sp. oral clone GI032
AY349396



Prevotella sp. oral clone GI059
AY349397



Prevotella sp. oral clone GU027
AY349398



Prevotella sp. oral clone HF050
AY349399



Prevotella sp. oral clone ID019
AY349400



Prevotella sp. oral clone IDR_CEC_0055
AY550997



Prevotella sp. oral clone IK053
AY349401



Prevotella sp. oral clone IK062
AY349402



Prevotella sp. oral clone P4PB_83 P2
AY207050



Prevotella sp. oral taxon 292
GQ422735



Prevotella sp. oral taxon 299
ACWZ01000026



Prevotella sp. oral taxon 300
GU409549



Prevotella sp. oral taxon 302
ACZK01000043



Prevotella sp. oral taxon 310
GQ422737



Prevotella sp. oral taxon 317
ACQH01000158



Prevotella sp. oral taxon 472
ACZS01000106



Prevotella sp. oral taxon 781
GQ422744



Prevotella sp. oral taxon 782
GQ422745



Prevotella sp. oral taxon F68
HM099652



Prevotella sp. oral taxon G60
GU432133



Prevotella sp. oral taxon G70
GU432179



Prevotella sp. oral taxon G71
GU432180



Prevotella sp. SEQ053
JN867222



Prevotella sp. SEQ065
JN867234



Prevotella sp. SEQ072
JN867238



Prevotella sp. SEQ116
JN867246



Prevotella sp. SG12
GU561343



Prevotella sp. sp24
AB003384



Prevotella sp. sp34
AB003385



Prevotella
stercorea

AB244774



Prevotella
tannerae

ACIJ02000018



Prevotella
timonensis

ADEF01000012



Prevotella
veroralis

ACVA01000027



Prevotella
jejuni, Prevotellaaurantiaca,




Prevotella
baroniae, Prevotellacolorans,




Prevotella
corporis, Prevotelladentasini,




Prevotella
enoeca, Prevotellafalsenii, Prevotella




fusca, Prevotellaheparinolytica, Prevotella




loescheii, Prevotellamultisaccharivorax,




Prevotella
nanceiensis,
Prevotella
oryzae,




Prevotella
paludivivens, Prevotellapleuritidis,




Prevotella
ruminicola, Prevotella




saccharolytica, Prevotellascopos, Prevotella




shahii, Prevotellazoogleoformans




Prevotellaceae bacterium P4P_62 P1
AY207061



Prochlorococcus
marinus

CP000551



Propionibacteriaceae bacterium NML 02_0265
EF599122



Propionibacterium
acidipropionici

NC_019395



Propionibacterium
acnes

ADJM01000010



Propionibacterium
avidum

AJ003055



Propionibacterium
freudenreichii

NR_036972



Propionibacterium
granulosum

FJ785716



Propionibacterium
jensenii

NR_042269



Propionibacterium
propionicum

NR_025277



Propionibacterium sp. 434_HC2
AFIL01000035



Propionibacterium sp. H456
AB177643



Propionibacterium sp. LG
AY354921



Propionibacterium sp. oral taxon 192
GQ422728



Propionibacterium sp. S555a
AB264622



Propionibacterium
thoenii

NR_042270



Proteus
mirabilis

ACLE01000013



Proteus
penneri

ABVP01000020



Proteus sp. HS7514
DQ512963



Proteus
vulgaris

AJ233425



Providencia
alcalifaciens

ABXW01000071



Providencia
rettgeri

AM040492



Providencia
rustigianii

AM040489



Providencia
stuartii

AF008581



Pseudoclavibacter sp. Timone
FJ375951



Pseudoflavonifractor
capillosus

AY136666


Pseudomonas aeruginosa
AABQ07000001



Pseudomonas
fluorescens

AY622220



Pseudomonas
gessardii

FJ943496



Pseudomonas
mendocina

AAUL01000021



Pseudomonas
monteilii

NR_024910



Pseudomonas
poae

GU188951



Pseudomonas
pseudoalcaligenes

NR_037000



Pseudomonas
putida

AF094741



Pseudomonas sp. 2_1_26
ACWU01000257



Pseudomonas sp. G1229
DQ910482



Pseudomonas sp. NP522b
EU723211



Pseudomonas
stutzeri

AM905854



Pseudomonas
tolaasii

AF320988



Pseudomonas
viridiflava

NR_042764



Pseudoramibacter
alactolyticus

AB036759



Psychrobacter
arcticus

CP000082



Psychrobacter
cibarius

HQ698586



Psychrobacter
cryohalolentis

CP000323



Psychrobacter
faecalis

HQ698566



Psychrobacter
nivimaris

HQ698587



Psychrobacter
pulmonis

HQ698582



Psychrobacter sp. 13983
HM212668



Pyramidobacter
piscolens

AY207056



Ralstonia
pickettii

NC_010682



Ralstonia sp. 5_7_47FAA
ACUF01000076



Raoultella
ornithinolytica

AB364958



Raoultella
planticola

AF129443



Raoultella
terrigena

NR_037085



Rhodobacter sp. oral taxon C30
HM099648



Rhodobacter
sphaeroides

CP000144



Rhodococcus
corynebacterioides

X80615



Rhodococcus
equi

ADNW01000058



Rhodococcus
erythropolis

ACNO01000030



Rhodococcus
fascians

NR_037021



Rhodopseudomonas
palustris

CP000301



Rickettsia
akari

CP000847



Rickettsia
conorii

AE008647



Rickettsia
prowazekii

M21789



Rickettsia
rickettsii

NC_010263



Rickettsia
slovaca

L36224



Rickettsia
typhi

AE017197



Robinsoniella
peoriensis

AF445258



Roseburia
cecicola

GU233441



Roseburia
faecalis

AY804149



Roseburia
faecis

AY305310



Roseburia
hominis

AJ270482



Roseburia
intestinalis

FP929050



Roseburia
inulinivorans

AJ270473



Roseburia sp. 11SE37
FM954975



Roseburia sp. 11SE38
FM954976



Roseiflexus
castenholzii

CP000804



Roseomonas
cervicalis

ADVL01000363



Roseomonas
mucosa

NR_028857



Roseomonas sp. NML94_0193
AF533357



Roseomonas sp. NML97_0121
AF533359



Roseomonas sp. NML98_0009
AF533358



Roseomonas sp. NML98_0157
AF533360



Rothia
aeria

DQ673320



Rothia
dentocariosa

ADDW01000024



Rothia
mucilaginosa

ACVO01000020



Rothia
nasimurium

NR_025310



Rothia sp. oral taxon 188
GU470892



Ruminobacter
amylophilus

NR_026450



Ruminococcaceae bacterium D16
ADDX01000083



Ruminococcus
albus

AY445600



Ruminococcus
bromii

EU266549



Ruminococcus
callidus

NR_029160



Ruminococcus
champanellensis

FP929052



Ruminococcus
flavefaciens

NR_025931



Ruminococcus
gnavus

X94967



Ruminococcus
hansenii

M59114



Ruminococcus
lactaris

ABOU02000049



Ruminococcus
obeum

AY169419



Ruminococcus sp. 18P13
AJ515913



Ruminococcus sp. 5_1_39BFAA
ACII01000172



Ruminococcus sp. 9SE51
FM954974



Ruminococcus sp. ID8
AY960564



Ruminococcus sp. K_1
AB222208



Ruminococcus
torques

AAVP02000002



Saccharomonospora
viridis

X54286



Salmonella
bongori

NR_041699



Salmonella
enterica

NC_011149



Salmonella
enterica

NC_011205



Salmonella
enterica

DQ344532



Salmonella
enterica

ABEH02000004



Salmonella
enterica

ABAK02000001



Salmonella
enterica

NC_011080



Salmonella
enterica

EU118094



Salmonella
enterica

NC_011094



Salmonella
enterica

AE014613



Salmonella
enterica

ABFH02000001



Salmonella
enterica

ABEM01000001



Salmonella
enterica

ABAM02000001



Salmonella
typhimurium

DQ344533



Salmonella
typhimurium

AF170176



Sarcina
ventriculi

NR_026146



Scardovia
inopinata

AB029087



Scardovia
wiggsiae

AY278626



Segniliparus
rotundus

CP001958



Segniliparus
rugosus

ACZI01000025



Selenomonas
artemidis

HM596274



Selenomonas
dianae

GQ422719



Selenomonas
flueggei

AF287803



Selenomonas genomosp. C1
AY278627



Selenomonas genomosp. C2
AY278628



Selenomonas genomosp. P5
AY341820



Selenomonas genomosp. P6 oral clone
DQ003636


MB3_C41




Selenomonas genomosp. P7 oral clone
DQ003627


MB5_C08




Selenomonas genomosp. P8 oral clone
DQ003628


MB5_P06




Selenomonas
infelix

AF287802



Selenomonas
noxia

GU470909



Selenomonas
ruminantium

NR_075026



Selenomonas sp. FOBRC9
HQ616378



Selenomonas sp. oral clone FT050
AY349403


Selenomonas sp. oral clone GI064
AY349404



Selenomonas sp. oral clone GT010
AY349405



Selenomonas sp. oral clone HU051
AY349406



Selenomonas sp. oral clone IK004
AY349407



Selenomonas sp. oral clone IQ048
AY349408



Selenomonas sp. oral clone JI021
AY349409



Selenomonas sp. oral clone JS031
AY349410



Selenomonas sp. oral clone OH4A
AY947498



Selenomonas sp. oral clone P2PA_80 P4
AY207052



Selenomonas sp. oral taxon 137
AENV01000007



Selenomonas sp. oral taxon 149
AEEJ01000007



Selenomonas
sputigena

ACKP02000033



Serratia
fonticola

NR_025339



Serratia
liquefaciens

NR_042062



Serratia
marcescens

GU826157



Serratia
odorifera

ADBY01000001



Serratia
proteamaculans

AAUN01000015



Shewanella
putrefaciens

CP002457



Shigella
boydii

AAKA01000007



Shigella
dysenteriae

NC_007606



Shigella
flexneri

AE005674



Shigella
sonnei

NC_007384



Shuttleworthia
satelles

ACIP02000004



Shuttleworthia sp. MSX8B
HQ616383



Shuttleworthia sp. oral taxon G69
GU432167



Simonsiella
muelleri

ADCY01000105



Slackia
equolifaciens

EU377663



Slackia
exigua

ACUX01000029



Slackia
faecicanis

NR_042220



Slackia
heliotrinireducens

NR_074439



Slackia
isoflavoniconvertens

AB566418



Slackia
piriformis

AB490806



Slackia sp. NATTS
AB505075



Solobacterium
moorei

AECQ01000039



Sphingobacterium
faecium

NR_025537



Sphingobacterium
mizutaii

JF708889



Sphingobacterium
multivorum

NR_040953



Sphingobacterium
spiritivorum

ACHA02000013



Sphingomonas
echinoides

NR_024700



Sphingomonas sp. oral clone FI012
AY349411



Sphingomonas sp. oral clone FZ016
AY349412



Sphingomonas sp. oral taxon A09
HM099639



Sphingomonas sp. oral taxon F71
HM099645



Sphingopyxis
alaskensis

CP000356



Spiroplasma
insolitum

NR_025705



Sporobacter
termitidis

NR_044972



Sporolactobacillus
inulinus

NR_040962



Sporolactobacillus
nakayamae

NR_042247



Sporosarcina
newyorkensis

AFPZ01000142



Sporosarcina sp. 2681
GU994081



Staphylococcaceae bacterium NML 92_0017
AY841362



Staphylococcus
aureus

CP002643



Staphylococcus
auricularis

JQ624774



Staphylococcus
capitis

ACFR01000029



Staphylococcus
caprae

ACRH01000033



Staphylococcus
carnosus

NR_075003



Staphylococcus
cohnii

JN175375



Staphylococcus
condimenti

NR_029345



Staphylococcus
epidermidis

ACHE01000056



Staphylococcus
equorum

NR_027520



Staphylococcus
fleurettii

NR_041326



Staphylococcus
haemolyticus

NC_007168



Staphylococcus
hominis

AM157418



Staphylococcus
lugdunensis

AEQA01000024



Staphylococcus
pasteuri

FJ189773



Staphylococcus
pseudintermedius

CP002439



Staphylococcus
saccharolyticus

NR_029158



Staphylococcus
saprophyticus

NC_007350



Staphylococcus
sciuri

NR_025520



Staphylococcus sp. clone bottae7
AF467424



Staphylococcus sp. H292
AB177642



Staphylococcus sp. H780
AB177644



Staphylococcus
succinus

NR_028667



Staphylococcus
vitulinus

NR_024670



Staphylococcus
warneri

ACPZ01000009



Staphylococcus
xylosus

AY395016



Stenotrophomonas
maltophilia

AAVZ01000005



Stenotrophomonas sp. FG_6
EF017810



Streptobacillus
moniliformis

NR_027615



Streptococcus
agalactiae

AAJO01000130



Streptococcus
alactolyticus

NR_041781



Streptococcus
anginosus

AECT01000011



Streptococcus
australis

AEQR01000024



Streptococcus
bovis

AEEL01000030



Streptococcus
canis

AJ413203



Streptococcus
constellatus

AY277942



Streptococcus
cristatus

AEVC01000028



Streptococcus
downei

AEKN01000002



Streptococcus
dysgalactiae

AP010935



Streptococcus
equi

CP001129



Streptococcus
equinus

AEVB01000043



Streptococcus
gallolyticus

FR824043



Streptococcus genomosp. C1
AY278629



Streptococcus genomosp. C2
AY278630



Streptococcus genomosp. C3
AY278631



Streptococcus genomosp. C4
AY278632



Streptococcus genomosp. C5
AY278633



Streptococcus genomosp. C6
AY278634



Streptococcus genomosp. C7
AY278635



Streptococcus genomosp. C8
AY278609



Streptococcus
gordonii

NC_009785



Streptococcus
infantarius

ABJK02000017



Streptococcus
infantis

AFNN01000024



Streptococcus
intermedius

NR_028736



Streptococcus
lutetiensis

NR_037096



Streptococcus
massiliensis

AY769997



Streptococcus
milleri

X81023



Streptococcus
mitis

AM157420



Streptococcus
mutans

AP010655



Streptococcus
oligofermentans

AY099095



Streptococcus
oralis

ADMV01000001



Streptococcus
parasanguinis

AEKM01000012



Streptococcus
pasteurianus

AP012054



Streptococcus
peroris

AEVF01000016



Streptococcus
pneumoniae

AE008537



Streptococcus
porcinus

EF121439



Streptococcus
pseudopneumoniae

FJ827123



Streptococcus
pseudoporcinus

AENS01000003



Streptococcus
pyogenes

AE006496



Streptococcus
ratti

X58304


Streptococcus salivarius
AGBV01000001



Streptococcus
sanguinis

NR_074974



Streptococcus
sinensis

AF432857



Streptococcus sp. 16362
JN590019



Streptococcus sp. 2_1_36FAA
ACOI01000028



Streptococcus sp. 2285_97
AJ131965



Streptococcus sp. 69130
X78825



Streptococcus sp. AC15
HQ616356



Streptococcus sp. ACS2
HQ616360



Streptococcus sp. AS20
HQ616366



Streptococcus sp. BS35a
HQ616369



Streptococcus sp. C150
ACRI01000045



Streptococcus sp. CM6
HQ616372



Streptococcus sp. CM7
HQ616373



Streptococcus sp. ICM10
HQ616389



Streptococcus sp. ICM12
HQ616390



Streptococcus sp. ICM2
HQ616386



Streptococcus sp. ICM4
HQ616387



Streptococcus sp. ICM45
HQ616394



Streptococcus sp. M143
ACRK01000025



Streptococcus sp. M334
ACRL01000052



Streptococcus sp. OBRC6
HQ616352



Streptococcus sp. oral clone ASB02
AY923121



Streptococcus sp. oral clone ASCA03
DQ272504



Streptococcus sp. oral clone ASCA04
AY923116



Streptococcus sp. oral clone ASCA09
AY923119



Streptococcus sp. oral clone ASCB04
AY923123



Streptococcus sp. oral clone ASCB06
AY923124



Streptococcus sp. oral clone ASCC04
AY923127



Streptococcus sp. oral clone ASCC05
AY923128



Streptococcus sp. oral clone ASCC12
DQ272507



Streptococcus sp. oral clone ASCD01
AY923129



Streptococcus sp. oral clone ASCD09
AY923130



Streptococcus sp. oral clone ASCD10
DQ272509



Streptococcus sp. oral clone ASCE03
AY923134



Streptococcus sp. oral clone ASCE04
AY953253



Streptococcus sp. oral clone ASCE05
DQ272510



Streptococcus sp. oral clone ASCE06
AY923135



Streptococcus sp. oral clone ASCE09
AY923136



Streptococcus sp. oral clone ASCE10
AY923137



Streptococcus sp. oral clone ASCE12
AY923138



Streptococcus sp. oral clone ASCF05
AY923140



Streptococcus sp. oral clone ASCF07
AY953255



Streptococcus sp. oral clone ASCF09
AY923142



Streptococcus sp. oral clone ASCG04
AY923145



Streptococcus sp. oral clone BW009
AY005042



Streptococcus sp. oral clone CH016
AY005044



Streptococcus sp. oral clone GK051
AY349413



Streptococcus sp. oral clone GM006
AY349414



Streptococcus sp. oral clone P2PA_41 P2
AY207051



Streptococcus sp. oral clone P4PA_30 P4
AY207064



Streptococcus sp. oral taxon 071
AEEP01000019



Streptococcus sp. oral taxon G59
GU432132



Streptococcus sp. oral taxon G62
GU432146



Streptococcus sp. oral taxon G63
GU432150



Streptococcus sp. SHV515
Y07601



Streptococcus
suis

FM252032



Streptococcus
thermophilus

CP000419



Streptococcus
uberis

HQ391900



Streptococcus
urinalis

DQ303194



Streptococcus
vestibularis

AEKO01000008



Streptococcus
viridans

AF076036



Streptomyces
albus

AJ697941



Streptomyces
griseus

NR_074787



Streptomyces sp. 1 AIP_2009
FJ176782



Streptomyces sp. SD 511
EU544231



Streptomyces sp. SD 524
EU544234



Streptomyces sp. SD 528
EU544233



Streptomyces sp. SD 534
EU544232



Streptomyces
thermoviolaceus

NR_027616



Subdoligranulum variabile
AJ518869



Succinatimonas
hippei

AEVO01000027



Sutterella
morbirenis

AJ832129



Sutterella
parvirubra

AB300989



Sutterella
sanguinus

AJ748647



Sutterella sp. YIT 12072
AB491210



Sutterella
stercoricanis

NR_025600



Sutterella
wadsworthensis

ADMF01000048



Synergistes genomosp. C1
AY278615



Synergistes sp. RMA 14551
DQ412722



Synergistetes bacterium ADV897
GQ258968



Synergistetes bacterium LBVCM1157
GQ258969



Synergistetes bacterium oral taxon 362
GU410752



Synergistetes bacterium oral taxon D48
GU430992



Syntrophococcus
sucromutans

NR_036869



Syntrophomonadaceae genomosp. P1
AY341821



Tannerella
forsythia

CP003191



Tannerella sp. 6_1_58FAA_CTI
ACWX01000068



Tatlockia
micdadei

M36032



Tatumella
ptyseos

NR_025342



Tessaracoccus sp. oral taxon F04
HM099640



Tetragenococcus
halophilus

NR_075020



Tetragenococcus
koreensis

NR_043113



Thermoanaerobacter
pseudethanolicus

CP000924



Thermobifida
fusca

NC_007333



Thermofilum
pendens

X14835



Thermus
aquaticus

NR_025900



Tissierella
praeacuta

NR_044860



Trabulsiella
guamensis

AY373830


Treponema denticola
ADEC01000002



Treponema genomosp. P1
AY341822



Treponema genomosp. P4 oral clone MB2_G19
DQ003618



Treponema genomosp. P5 oral clone MB3_P23
DQ003624



Treponema genomosp. P6 oral clone MB4_G11
DQ003625



Treponema
lecithinolyticum

NR_026247



Treponema
pallidum

CP001752



Treponema
parvum

AF302937



Treponema
phagedenis

AEFH01000172



Treponema
putidum

AJ543428



Treponema
refringens

AF426101



Treponema
socranskii

NR_024868



Treponema sp. 6:H:D15A_4
AY005083



Treponema sp. clone DDKL_4
Y08894



Treponema sp. oral clone JU025
AY349417



Treponema sp. oral clone JU031
AY349416



Treponema sp. oral clone P2PB_53P3
AY207055



Treponema sp. oral taxon 228
GU408580



Treponema sp. oral taxon 230
GU408603



Treponema sp. oral taxon 231
GU408631



Treponema sp. oral taxon 232
GU408646



Treponema sp. oral taxon 235
GU408673



Treponema sp. oral taxon 239
GU408738



Treponema sp. oral taxon 247
GU408748



Treponema sp. oral taxon 250
GU408776



Treponema sp. oral taxon 251
GU408781



Treponema sp. oral taxon 254
GU408803



Treponema sp. oral taxon 265
GU408850



Treponema sp. oral taxon 270
GQ422733



Treponema sp. oral taxon 271
GU408871



Treponema sp. oral taxon 508
GU413616



Treponema sp. oral taxon 518
GU413640



Treponema sp. oral taxon G85
GU432215



Treponema sp. ovine footrot
AJ010951



Treponema
vincentii

ACYH01000036



Tropheryma
whipplei

BX251412



Trueperella
pyogenes

NR_044858



Tsukamurella
paurometabola

X80628



Tsukamurella
tyrosinosolvens

AB478958



Turicibacter
sanguinis

AF349724



Ureaplasma
parvum

AE002127



Ureaplasma
urealyticum

AAYN01000002



Ureibacillus
composti

NR_043746



Ureibacillus
suwonensis

NR_043232



Ureibacillus
terrenus

NR_025394



Ureibacillus
thermophilus

NR_043747



Ureibacillus
thermosphaericus

NR_040961



Vagococcus
fluvialis

NR_026489



Veillonella
atypica

AEDS01000059



Veillonella
dispar

ACIK02000021



Veillonella genomosp. P1 oral clone MB5_P17
DQ003631



Veillonella
montpellierensis

AF473836



Veillonella
parvula

ADFU01000009



Veillonella sp. 3_1_44
ADCV01000019



Veillonella sp. 6_1_27
ADCW01000016



Veillonella sp. ACP1
HQ616359



Veillonella sp. AS16
HQ616365



Veillonella sp. BS32b
HQ616368



Veillonella sp. ICM51a
HQ616396



Veillonella sp. MSA12
HQ616381



Veillonella sp. NVG 100cf
EF108443



Veillonella sp. OK11
JN695650



Veillonella sp. oral clone ASCA08
AY923118



Veillonella sp. oral clone ASCB03
AY923122



Veillonella sp. oral clone ASCG01
AY923144



Veillonella sp. oral clone ASCG02
AY953257



Veillonella sp. oral clone OH1A
AY947495



Veillonella sp. oral taxon 158
AENU01000007



Veillonellaceae bacterium oral taxon 131
GU402916



Veillonellaceae bacterium oral taxon 155
GU470897



Vibrio
cholerae

AAUR01000095



Vibrio
fluvialis

X76335



Vibrio
furnissii

CP002377



Vibrio
mimicus

ADAF01000001



Vibrio
parahaemolyticus

AAWQ01000116



Vibrio sp. RC341
ACZT01000024



Vibrio
vulnificus

AE016796



Victivallaceae bacterium NML 080035
FJ394915



Victivallis
vadensis

ABDE02000010



Virgibacillus
proomii

NR_025308



Weissella
beninensis

EU439435



Weissella
cibaria

NR_036924



Weissella
confusa

NR_040816



Weissella
hellenica

AB680902



Weissella
kandleri

NR_044659



Weissella
koreensis

NR_075058



Weissella
paramesenteroides

ACKU01000017



Weissella sp. KLDS 7.0701
EU600924



Wolinella
succinogenes

BX571657



Xanthomonadaceae bacterium NML 03_0222
EU313791



Xanthomonas
campestris

EF101975



Xanthomonas sp. kmd_489
EU723184



Xenophilus
aerolatus

JN585329



Yersinia
aldovae

AJ871363



Yersinia
aleksiciae

AJ627597



Yersinia
bercovieri

AF366377



Yersinia
enterocolitica

FR729477



Yersinia
frederiksenii

AF366379



Yersinia
intermedia

AF366380



Yersinia
kristensenii

ACCA01000078



Yersinia
mollaretii

NR_027546



Yersinia
pestis

AE013632



Yersinia
pseudotuberculosis

NC_009708



Yersinia
rohdei

ACCD01000071



Yokenella
regensburgei

AB273739



Zimmermannella
bifida

AB012592



Zymomonas
mobilis

NR_074274
















TABLE 2







Exemplary Oncophilic Bacteria









Genera
Species
Tumor Association






Mycoplasma


hyorhinis

Gastric Carcinoma



Propionibacterium


Acnes

Prostate Cancer



Mycoplasma


genitalium

Prostate Cancer



Methylophilus

sp.
Prostate Cancer



Chlamydia


trachomatis

Prostate Cancer



Helicobacter


pylori

Gastric MALT



Listeria


welshimeri

Renal Cancer



Streptococcus


pneumoniae

Lymphoma and Leukemia



Haemophilus


influenzae

Lymphoma and Leukemia



Staphylococcus


aureus

Breast Cancer



Listeria


monocytogenes

Breast Cancer



Methylobacterium


radiotolerans

Breast Cancer



Shingomonas


yanoikuyae

breast Cancer



Fusobacterium

sp
Larynx cancer



Provetelis

sp
Larynx cancer



streptococcus


pneumoniae

Larynx cancer



Gemella

sp
Larynx cancer



Bordetella


Pertussis

Larynx cancer



Corumebacterium


tuberculosteraricum

Oral squamous cell carcinoma



Micrococcus


luteus

Oral squamous cell carcinoma



Prevotella


melaninogenica

Oral squamous cell carcinoma



Exiguobacterium


oxidotolerans

Oral squamous cell carcinoma



Fusobacterium


naviforme

Oral squamous cell carcinoma



Veillonella


parvula

Oral squamous cell carcinoma



Streptococcus


salivarius

Oral squamous cell carcinoma



Streptococcus


mitis/oralis
Oral squamous cell carcinoma



veillonella


dispar

Oral squamous cell carcinoma



Peptostreptococcus


stomatis

Oral squamous cell carcinoma



Streptococcus


gordonii

Oral squamous cell carcinoma



Gemella


Haemolysans

Oral squamous cell carcinoma



Gemella


morbillorum

Oral squamous cell carcinoma



Johnsonella


ignava

Oral squamous cell carcinoma



Streptococcus


parasanguins

Oral squamous cell carcinoma



Granulicatella


adiacens

Oral squamous cell carcinoma



Mycobacteria


marinum

lung infection



Campylobacter


concisus

Barrett's Esophagus



Campylobacter


rectus

Barrett's Esophagus



Oribacterium

sp
Esophageal adenocarcinoma



Catonella

sp
Esophageal adenocarcinoma



Peptostreptococcus

sp
Esophageal adenocarcinoma



Eubacterium

sp
Esophageal adenocarcinoma



Dialister

sp
Esophageal adenocarcinoma



Veillonella

sp
Esophageal adenocarcinoma



Anaeroglobus

sp
Esophageal adenocarcinoma



Megasphaera

sp
Esophageal adenocarcinoma



Atoppbium

sp
Esophageal adenocarcinoma



Solobacterium

sp
Esophageal adenocarcinoma



Rothia

sp
Esophageal adenocarcinoma



Actinomyces

sp
Esophageal adenocarcinoma



Fusobacterium

sp
Esophageal adenocarcinoma



Sneathia

sp
Esophageal adenocarcinoma



Leptotrichia

sp
Esophageal adenocarcinoma



Capnocytophaga

sp
Esophageal adenocarcinoma



Prevotella

sp
Esophageal adenocarcinoma



Porphyromonas

sp
Esophageal adenocarcinoma



Campylobacter

sp
Esophageal adenocarcinoma



Haemophilus

sp
Esophageal adenocarcinoma



Neisseria

sp
Esophageal adenocarcinoma


TM7
sp
Esophageal adenocarcinoma



Granulicatella

sp
Esophageal adenocarcinoma



Variovorax

sp
Psuedomyxoma Peritonei



Escherichia


Shigella

Psuedomyxoma Peritonei



Pseudomonas

sp
Psuedomyxoma Peritonei



Tessaracoccus

sp
Psuedomyxoma Peritonei



Acinetobacter

sp
Psuedomyxoma Peritonei



Helicobacter


hepaticus

Breast cancer



Chlamydia


psittaci

MALT lymphoma



Borrelia


burgdorferi

B cell lymphoma skin



Escherichia


Coli NC101
Colorectal Cancer



Salmonella


typhimurium

Tool



Eterococcus


faecalis

blood



Streptococcus


mitis

blood



Streptococcus


sanguis

blood



Streptococcus


anginosus

blood


Streptococcus

salvarius

blood



Staphylococcus


epidermidis

blood



Streptococcus


gallolyticus

Colorectal Cancer



Campylobacter


showae CC57C
Colorectal Cancer



Leptotrichia

sp
Colorectal Cancer









In certain embodiments the EVs described herein areobtained from obligate anaerobic bacteria. Examples of obligate anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella spp.), gram-positive cocci (primarily Peptostreptococcus spp.), gram-positive spore-forming (Clostridium spp.), non-spore-forming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Bifidobacterium spp.), and gram-negative cocci (mainly Veillonella spp.). In some embodiments, the obligate anearoic bacteria are of a genus selected from Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.


In some embodiments, the EVs described herein are obtained from bacterium of a genus selected from Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcusa.


In some embodiments, the bacteria and/or EVs described herein are of a species selected from Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Veillonella tobetsuensis.


In some embodiments, the EVs and/or bacteria described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety. In some embodiments, the therapeutic moiety is a cancer-specific moiety. In some embodiments, the cancer-specific moiety has binding specificity for a cancer cell (e.g., has binding specificity for a cancer-specific antigen). In some embodiments, the cancer-specific moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In some embodiments, the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (e.g., by having binding specificity for a cancer-specific antigen). In some embodiments, the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the first part has binding specificity for the EV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the first and/or second part comprises an antibody or antigen binding fragment thereof. In some embodiments, the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the cancer-specific moiety with the EVs (either in combination or in separate administrations) increases the targeting of the EVs to the cancer cells.


In some embodiments, the EVs described herein is modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead). In some embodiments, the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV. In some embodiments, the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the EV-binding moiety has binding specificity for the EV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the EV-binding moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the EVs (either together or in separate administrations) can be used to increase the targeting of the EVs to cancer calls and/or a part of a subject where cancer cells are present.


Production of EVs


In certain aspects, the EVs described herein can be prepared using any method known in the art.


In some embodiments, the EVs are prepared without an EV purification step. For example, in some embodiments, bacteria comprising the EVs described herein are killed using a method that leaves the bacterial EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation.


In some embodiments, the EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art. In some embodiments EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLOS ONE. 6(3):e17629 (2011) or G. Norheim, et al. PLOS ONE. 10(9): e0134353 (2015), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000×g for 30 min at 4° C.). In some embodiments, the culture supernatants are then passed through filter to exclude intact bacterial cells (e.g., a 0.22 μm filter). In some embodiments, filtered supernatants are centrifuged to pellet bacterial EVs (e.g., at 100,000-150,000×g for 1-3 hours at 4° C.). In some embodiments, the EVs are further purified by resuspending the resulting EV pellets (e.g., in PBS), and applying the resuspended EVs to sucrose gradient (e.g., a 30-60% discontinuous sucrose gradient), followed by centrifugation (e.g., at 200,000×g for 20 hours at 4° C.). EV bands can be collected, washed with (e.g., with PBS), and centrifuged to pellet the EVs (e.g., at 150,000×g for 3 hours at 4° C.). The purified EVs can be stored, for example, at −80° C. until use. In some embodiments, the EVs are further purified by treatment with DNase and/or proteinase K


For example, in some embodiments, cultures of bacteria disclosed herein can be centrifuged at 11,000×g for 20-40 min at 4° C. to pellet bacteria. Culture supernatants may be passed through a 0.22 μm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4° C. Precipitations can be incubated at 4° C. for 8-48 hours and then centrifuged at 11,000×g for 20-40 min at 4° C. The resulting pellets contain bacterial EVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000-200,000×g for 1-16 hours at 4° ° C. The pellet of this centrifugation contains bacterial EVs and other debris. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight>50 or 100 kDa.


Alternatively, EVs can be obtained from bacterial cultures continuously during growth, or at selected time points during growth, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., EVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 um and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.


EVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24 hours at 4° ° C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24 hours at 4° C.


In some embodiments, to confirm sterility and isolation of the EV preparations, EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.


In some embodiments, for preparation of EVs used for in vivo injections, purified EVs are processed as described previously (G. Norheim, et al. PLOS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 g/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).


In certain embodiments, to make samples compatible with further testing (e.g. to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation (200,000×g, ≥3 hours, 4° C.) and resuspension.


In some embodiments, the sterility of the EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.


In some embodiments select EVs are isolated and enriched by chromatography and binding surface moieties on EVs. In other embodiments, select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.


Pharmaceutical Compositions


In certain embodiments, the methods provided herein are pharmaceutical compositions comprising EVs and/or bacteria provided herein (e.g., an EV composition). In some embodiments, the EV composition comprises an EV and/or a combination of EVs described herein and a pharmaceutically acceptable carrier.


In some embodiments, the pharmaceutical compositions comprise EVs substantially or entirely free of bacteria. In some embodiments, the pharmaceutical compositions comprise both EVs and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In certain embodiments, the pharmaceutical compositions comprise bacteria that is substantially or entirely free of EVs. In some embodiments, the pharmaceutical compositions comprise EVs and/or bacteria from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the bacteria strains or species listed in Table 1 and/or Table 2.


In some embodiments, the pharmaceutical composition comprises at least 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles.


In some embodiments, the pharmaceutical composition comprises about 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles.


In certain embodiments, the pharmaceutical composition comprises a certain ratio of bacteria particles to EV particles. The number of bacteria particles can be based on actual particle number or (if the bacteria is live) the number of CFUs. The particle number can be established by combining a set number of purified EVs with a set number of purified bacterium, by modifying the growth conditions under which the bacteria are cultured, or by modifying the bacteria itself to produce more or fewer EVs.


In some embodiments, to quantify the numbers of EVs and/or bacteria present in a bacterial sample, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the vesicles and bacteria and count their relative numbers. Alternatively, combinations of nanoparticle tracking analysis (NTA), Coulter counting, and dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 μm. The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria in a given sample. Coulter counting reveals the numbers of particles with diameters of 0.7-10 um. NTA reveals the numbers of particles with diameters of 50-1400 nm. For most bacterial samples, the Coulter counter alone can reveal the number of bacteria in a sample. EVs are 20-250 nm in diameter. NTA will allow us to count the numbers of particles that are 50-250 nm in diameter. DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm-3 um.


In some embodiments, the pharmaceutical composition comprises no more than 1 bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 EV particles.


In some embodiments, the pharmaceutical composition comprises at least 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium.


In some embodiments, the pharmaceutical composition comprises about 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium. In some embodiments, the pharmaceutical composition comprises no more than 1 EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, and/or 1×1012 bacterium.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are EVs.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are bacteria.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are EVs.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are bacteria.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are EVs.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are bacteria.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is EV protein.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is bacteria protein.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is EV protein.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is bacteria protein.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is EV protein.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is bacteria protein.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are EV lipids.


In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are bacteria lipids.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are EV lipids.


In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are bacteria lipids.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are EV lipids.


In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are bacteria lipids.


In some embodiments, the EVs in the pharmaceutical composition are purified from one or more other bacterial components. In some embodiments, the pharmaceutical composition further comprises other bacterial components. In some embodiments, the pharmaceutical composition comprise bacteria cells.


In certain aspects, provided are pharmaceutical compositions for administration subjects. In some embodiments, the pharmaceutical compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format. In some embodiments, the the pharmaceutical compositions is combined with an adjuvant such as an immuno-adjuvant (e.g., STING agonists, TLR agonists, NOD agonists).


In some embodiments the composition comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnH2nOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.


In some embodiments the composition comprises at least one lipid. As used herein a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.


In some embodiments the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.


In some embodiments the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.


In some embodiments the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.


In some embodiments the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.


In some embodiments the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.


In some embodiments the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.


In some embodiments the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.


In some embodiments the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.


In some embodiments the composition comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.


In some embodiments, the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.


In some embodiments the composition is a food product for animals, including humans. The animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.


Therapeutic Agents


In certain aspects, the methods provided herein include the administration to a subject of a pharmaceutical composition described herein either alone or in combination with an additional therapeutic. In some embodiments, the additional therapeutic is an immunosuppressant, a steroid, cancer therapeutic.


In some embodiments the EV is administered to the subject before the therapeutic is administered (e.g., at least 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 before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before). In some embodiments the EV is administered to the subject after the therapeutic is administered (e.g., at least 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 after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, the EV and the therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other). In some embodiments, the subject is administered an antibiotic before the EV is administered to the subject (e.g., at least 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 before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before). In some embodiments, the subject is administered an antibiotic after the EV is administered to the subject (e.g., at least 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 before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, the EV and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).


In some embodiments, the additional therapeutic is a cancer therapeutic. In some embodiments, the cancer therapeutic is a chemotherapeutic agent. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegal1; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.


In some embodiments, the cancer therapeutic is a cancer immunotherapy agent. Immunotherapy refers to a treatment that uses a subject's immune system to treat cancer, e.g., checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy. Non-limiting examples of immunotherapies are checkpoint inhibitors include Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1), Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca, anti-PD-L1), and MPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumor vaccines, such as Gardail, CERVARIX® (Human Papillomavirus Bivalent (Types 16 and 18) Vaccine, Recombinant), BCG, sipulencel-T, Gp100:209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak® (aglatimagene besadenovec), PROSTVAC® (PSA-TRICOM or PROSTVAC®-V/R-TRICOM (rilimogene-galvacirepvec), Rindopepimul, E75 peptide acetate, IMA901, POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, and Tecemotide. Immunotherapy may be administered via injection (e.g., intravenously, intratumorally, subcutaneously, or into lymph nodes), but may also be administered orally, topically, or via aerosol. Immunotherapies may comprise adjuvants such as cytokines.


In some embodiments, the immunotherapy agent is an immune checkpoint inhibitor. Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.


In some embodiments, the methods provided herein include the administration of a pharmaceutical composition described herein in combination with one or more additional therapeutic. In some embodiments, the methods disclosed herein include the administration of two additional immunotherapy agents (e.g., immune checkpoint inhibitor). For example, the methods provided herein include the administration of a pharmaceutical composition described herein in combination with a PD-1 inhibitor and a CLTA-4 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor.


In some embodiments, the immunotherapy agent is an antibody or antigen binding fragment thereof that, for example, binds to a cancer-associated antigen. Examples of cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDHIA1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKIA1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AMLI fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pmel17, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHNI also known as CCDCl10, LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MCIR, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUCI, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAPI, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a. In some embodiments, the antigen is a neo-antigen.


In some embodiments, the immunotherapy agent is a cancer vaccine and/or a component of a cancer vaccine (e.g., an antigenic peptide and/or protein). The cancer vaccine can be a protein vaccine, a nucleic acid vaccine or a combination thereof. For example, in some embodiments, the cancer vaccine comprises a polypeptide comprising an epitope of a cancer-associated antigen. In some embodiments, the cancer vaccine comprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodes an epitope of a cancer-associated antigen. Examples of cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKIA1, CTAG1, CTAG2, cyclin D1, Cyclin-A1,dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AMLI fusion protein, EZH2, FGFS, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHNI also known as CCDCl10, LAGE-1, LDLR-fucosyltranferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MCIR, MCSP, mdm-2, MEL Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUCI, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAXS, PBF, pm1-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPPIR3B, PRAME, PRDXS, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGSS, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAPI, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a. In some embodiments, the antigen is a neo-antigen. In some embodiments, the cancer vaccine is administered with an adjuvant. Examples of adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG ODN DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, MONTANIDE®, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, cholera toxin (CT) and heat-labile toxin from enterotoxigenic Escherichia coli (LT) including derivatives of these (CTB, mmCT, CTA1-DD, LTB, LTK63, LTR72, dmLT) and trehalose dimycolate.


In some embodiments, the immunotherapy agent is an immune modulating protein to the subject. In some embodiments, the immune modulatory protein is a cytokine or chemokine. Examples of immune modulating proteins include, but are not limited to, B lymphocyte chemoattractant (“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon alpha (“IFN-alpha”), Interferon beta (“IFN-beta”) Interferon gamma (“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta (“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”), Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10 (“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12 (“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17A-F (“IL-17A-F”), Interleukin-18 (“IL-18”), Interleukin-21 (“IL-21”), Interleukin-22 (“IL-22”), Interleukin-23 (“IL-23”), Interleukin-33 (“IL-33”), Chemokine (C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1 beta”), Macrophage inflammatory protein-1-delta (“MIP-1 delta”), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated on Activation, Normal T cell Expressed and Secreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMP metallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor, lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta (“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growth factor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor (“EGF”), Epidermal growth factor receptor (“EGFR”), Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”), Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor (“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glial cell-derived neurotrophic factor (“GDNF”), Growth Hormone, Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growth factor (“HGF”), Insulin-like growth factor binding protein 1 (“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”), Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-like growth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factor binding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”), Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growth factor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4 (“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing comples (“SCF”), Stem cell factor receptor (“SCF R”), Transforming growth factor alpha (“TGFalpha”), Transforming growth factor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGF beta 3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C—X—C motif chemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”), Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”), Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C—X—C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3 alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derived factor-1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17 (“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromal lymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster of Differentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamily member 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster of Differentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40 Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”), Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosis factor receptor superfamily member 1 (“GITR”), Tumor necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R, Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associated lipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium (“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHC class I polypeptide-related sequence B (“MICB”), NRGI-betal, Beta-type platelet-derived growth factor receptor (“PDGF Rbeta”), Platelet endothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A virus cellular receptor 1 (“TIM-1”), Tumor necrosis factor receptor superfamily member IOC (“TRAIL R3”), Trappin protein transglutaminase binding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular cell adhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein (“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin, Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN, Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial cell adhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C, FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”), IL-13 RI, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal cell adhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1 (“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”), Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secreted frizzled-related protein 2 (“ShhN”), Sialic acid-binding immunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growth factor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumor necrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggering receptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelial growth factor C (“VEGF-C”), VEGFRIAdiponectin, Adipsin (“AND”), Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”), Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 (“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-like growth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2 (“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13 (“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin (“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9 (“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamily member 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogenetic protein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member 6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein, alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”), IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1 receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1, translocation-associated (Drosophila) (“Notch-1”), Nephroblastoma overexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1 (“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4, Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllike receptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member 10A (“TRAIL RI”), Transferrin (“TRF”), WIF-IACE-2, Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen 19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha (“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocyte colony-stimulating factor receptor (“GCSF R”), Serine protease hepsin (“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27 (“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V (“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST, Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily, member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNTI-inducible-signaling pathway protein 1 (“WISP-1”), and Receptor Activator of Nuclear Factor κ B (“RANK”).


In some embodiments, the cancer therapeutic agent is an anti-cancer compound. Exemplary anti-cancer compounds include, but are not limited to, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole (Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib (Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®), Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®), Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®), Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®), Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole (Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab (Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (Perjeta™), Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and 131I-tositumomab (Bexxar®), Trastuzumab (Herceptin®), Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).


Exemplary anti-cancer compounds that modify the function of proteins that regulate gene expression and other cellular functions (e.g., HDAC inhibitors, retinoid receptor ligants) are Vorinostat (Zolinza®), Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin (Panretin®), and Tretinoin (Vesanoid®).


Exemplary anti-cancer compounds that induce apoptosis (e.g., proteasome inhibitors, antifolates) are Bortezomib (Velcade®), Carfilzomib (Kyprolis™), and Pralatrexate (Folotyn®).


Exemplary anti-cancer compounds that increase anti-tumor immune response (e.g., anti CD20, anti CD52; anti-cytotoxic T-lymphocyte-associated antigen-4) are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab (Arzerra®), and Ipilimumab (Yervoy™).


Exemplary anti-cancer compounds that deliver toxic agents to cancer cells (e.g., anti-CD20-radionuclide fusions; IL-2-diphtheria toxin fusions; anti-CD30-monomethylauristatin E (MMAE)-fusions) are Tositumomab and 131I-tositumomab (Bexxar®) and Ibritumomab tiuxetan (Zevalin®), Denileukin diftitox (Ontak®), and Brentuximab vedotin (Adcetris®).


Other exemplary anti-cancer compounds are small molecule inhibitors and conjugates thereof of, e.g., Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGF receptor, Braf, MEK, CDK, and HSP90.


Exemplary platinum-based anti-cancer compounds include, for example, cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin. Other metal-based drugs suitable for treatment include, but are not limited to ruthenium-based compounds, ferrocene derivatives, titanium-based compounds, and gallium-based compounds.


In some embodiments, the cancer therapeutic is a radioactive moiety that comprises a radionuclide. Exemplary radionuclides include, but are not limited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, I-125, Eu-149, Os-189m, Sb-119, I-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, Tl-201, Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169, Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105, Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, I-131, Tb-161, As-77, Pt-197, Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109, Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr-142, Ir-194, In-114m/In-114, and Y-90.


In some embodiments, the cancer therapeutic is an antibiotic. For example, if the presence of a cancer-associated bacteria and/or a cancer-associated microbiome profile is detected according to the methods provided herein, antibiotics can be administered to eliminate the cancer-associated bacteria from the subject. “Antibiotics” broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability, or their spectrum of target microbe (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.) and these may be used to kill specific bacteria in specific areas of the host (“niches”) (Leekha, et al 2011. General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). In certain embodiments, antibiotics can be used to selectively target bacteria of a specific niche. In some embodiments, antibiotics known to treat a particular infection that includes a cancer niche may be used to target cancer-associated microbes, including cancer-associated bacteria in that niche. In other embodiments, antibiotics are administered after the bacterial treatment. In some embodiments, antibiotics are administered after the bacterial treatment to remove the engraftment.


In some aspects, antibiotics can be selected based on their bactericidal or bacteriostatic properties. Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., β-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones). Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis. Furthermore, while some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties. In certain treatment conditions, bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics. Thus, in certain embodiments, bactericidal and bacteriostatic antibiotics are not combined.


Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti-mycobacterial compounds, and combinations thereof.


Aminoglycosides include, but are not limited to Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin. Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.


Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.


Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.


Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Gram-positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.


Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. Selected Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin-resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.


Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.


Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus, and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.


Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.


Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g., against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis.


Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g., against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.


Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.


Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.


Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective, e.g., against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus, Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.


Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.


Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective, e.g., against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.


Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin. Quinolones/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria. Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.


Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), and Sulfonamidochrysoidine. Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.


Tetracyclines include, but are not limited to, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.


Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.


Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin, ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin, ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin, spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin, taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid, triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin, vemamycin, and virginiamycin.


In some embodiments, the additional therapeutic is an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal antiinflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof. Representative agents include, but are not limited to, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs (e.g., hydroxychloroquine and chloroquine), sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNF alpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra/Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Ilaris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/LymphoStat-B® (belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura-100, Oncoxin+Viusid, TwHF, Methoxsalen, Vitamin D—ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus (Prograf®), RADOOI, rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone, Curcumin (Longvida™), Rosuvastatin, Maraviroc, ramipnl, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists (Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.).


In some embodiments, the agent is an immunosuppressive agent. Examples of immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccines used for vaccination where the amount of an allergen is gradually increased), cytokine inhibitors, such as anti-IL-6 antibodies, TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept, iand combinations thereof.


Administration


In certain aspects, provided herein is a method of delivering a pharmaceutical composition described herein to a subject. In some embodiments of the methods provided herein, the pharmaceutical composition is administered in conjunction with the administration of an additional therapeutic. In some embodiments, the pharmaceutical composition comprises EVs and/or bacteria co-formulated with the additional therapeutic. In some embodiments, the pharmaceutical composition is co-administered with the additional therapeutic. In some embodiments, the additional therapeutic is administered to the subject before administration of the pharmaceutical composition (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments, the additional therapeutic is administered to the subject after administration of the pharmaceutical composition (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after). In some embodiments the same mode of delivery are used to deliver both the pharmaceutical composition and the additional therapeutic. In some embodiments different modes of delivery are used to administer the pharmaceutical composition and the additional therapeutic. For example, in some embodiments the pharmaceutical composition is administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, intramuscular and/or intratumoral injection).


In certain embodiments, the pharmaceutical compositions, dosage forms, and kits described herein can be administered in conjunction with any other conventional anti-cancer treatment, such as, for example, radiation therapy and surgical resection of the tumor. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the pharmaceutical compositions, dosage forms, and kits described herein.


The dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art. In the present methods, appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate. The dose of the pharmaceutical compositions described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like. For example, the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day. The effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.


In some embodiments, the dose administered to a subject is sufficient to prevent disease (e.g., autoimmune disease, inflammatory disease, metabolic disease, cancer), delay its onset, or slow or stop its progression. One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect.


Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose (“MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.


In accordance with the above, in therapeutic applications, the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and most preferably causing complete regression of the cancer.


Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of an pharmaceutical composition, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.


The time period between administrations can be any of a variety of time periods. The time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the EV from normal tissue. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month. In another example, the time period can be a function of the time period for a subject to clear the EV from normal tissue; for example, the time period can be more than the time period for a subject to clear the EV from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.


In some embodiments, the delivery of an additional therapeutic in combination with the pharmaceutical composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic.


The effective dose of an additional therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient. The effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. In general, an effective dose of an additional therapy will be the amount of the therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.


The toxicity of an additional therapy is the level of adverse effects experienced by the subject during and following treatment. Adverse events associated with additional therapy toxicity include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylasix, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia, dyspnea, edema, electrolyte imbalance, esophagitis, fatigue, loss of fertility, fever, flatulence, flushing, gastric reflux, gastroesophageal reflux disease, genital pain, granulocytopenia, gynecomastia, glaucoma, hair loss, hand-foot syndrome, headache, hearing loss, heart failure, heart palpitations, heartburn, hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia, hypophosphatemia, impotence, infection, injection site reactions, insomnia, iron deficiency, itching, joint pain, kidney failure, leukopenia, liver dysfunction, memory loss, menopause, mouth sores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis, neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds, numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia, pancytopenia, pericarditis, peripheral neuropathy, pharyngitis, photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria, pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapid heart beat, rectal bleeding, restlessness, rhinitis, seizures, shortness of breath, sinusitis, thrombocytopenia, tinnitus, urinary tract infection, vaginal bleeding, vaginal dryness, vertigo, water retention, weakness, weight loss, weight gain, and xerostomia. In general, toxicity is acceptable if the benefits to the subject achieved through the therapy outweigh the adverse events experienced by the subject due to the therapy.


Immune Disorders


In some embodiments, the methods and compositions described herein relate to the treatment or prevention a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease. In some embodiments, the disease or disorder is an inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis).


The methods described herein can be used to treat any subject in need thereof. As used herein, a “subject in need thereof” includes any subject that has a disease or disorder associated with a pathological immune response (e.g., an inflammatory bowel disease), as well as any subject with an increased likelihood of acquiring a such a disease or disorder.


The compositions described herein can be used, for example, as a pharmaceutical composition for preventing or treating (reducing, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory disease such as a TNF-mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease); a pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of immune cells.


In some embodiments, the methods provided herein are useful for the treatment of inflammation. In certain embodiments, the inflammation of any tissue and organs of the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.


Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons. Examples of such immune disorders, which may be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).


Ocular immune disorders refers to a immune disorder that affects any structure of the eye, including the eye lids. Examples of ocular immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis


Examples of nervous system immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia. Examples of inflammation of the vasculature or lymphatic system which may be treated with the methods and compositions described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.


Examples of digestive system immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis. Inflammatory bowel diseases include, for example, certain art-recognized forms of a group of related conditions. Several major forms of inflammatory bowel diseases are known, with Crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) the most common of these disorders. In addition, the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.


Examples of reproductive system immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.


The methods and compositions described herein may be used to treat autoimmune conditions having an inflammatory component. Such conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.


The methods and compositions described herein may be used to treat T-cell mediated hypersensitivity diseases having an inflammatory component. Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).


Other immune disorders which may be treated with the methods and compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopeniaurpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).


Metabolic Disorders


The methods and compositions described herein may be used to treat metabolic disorders and metabolic syndromes. Such conditions include, but are not limited to, Type II Diabetes, Encephalopathy, Tay-Sachs disease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), and Maple syrup urine disease. Accordingly, in certain embodiments provided herein are methods of treating metabolic diseases comprising administering to a subject a composition provided herein. In certain embodiments the metabolic disease is Type II Diabetes, Encephalopathy, Tay-Sachs disease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), or Maple syrup urine disease.


Cancer


In some embodiments, the methods and compositions described herein relate to the treatment of cancer. In some embodiments, any cancer can be treated using the methods described herein. Examples of cancers that may treated by methods and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.


In some embodiments, the methods and compositions provided herein relate to the treatment of a leukemia. The term “leukemia” is meant broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia.


In some embodiments, the methods and compositions provided herein relate to the treatment of a carcinoma. The term “carcinoma” refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and gives rise to metastases. Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.


In some embodiments, the methods and compositions provided herein relate to the treatment of a sarcoma. The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.


Additional exemplary neoplasias that can be treated using the methods and compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.


In some embodiments, the cancer treated is a melanoma. The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.


Particular categories of tumors that can be treated using methods and compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, plasmacytoma, colorectal cancer, and rectal cancer.


Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.


Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.


Other Diseases and Disorders


In some embodiments, the methods and compositions described herein relate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH).


In some embodiments, the methods and compositions described herein relate to the treatment of liver diseases. Such diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Non-Alcoholic Fatty Liver Disease, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease


The methods and compositions described herein may be used to treat neurodegenerative and neurological diseases. In certain embodiments, the neurodegenerative and/or neurological disease is Parkinson's disease, Alzheimer's disease, prion disease, Huntington's disease, motor neurone diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction.


Methods of Making Enhanced Bacteria


In certain aspects, provided herein are methods of making engineered bacteria for the production of the EVs described herein. In some embodiments, the engineered bacteria are modified to enhance certain desirable properties. For example, in some embodiments, the engineered bacteria are modified to increase production of EVs by the bacteria. In some embodiments, the engineered bacteria are modified to produce EVs with enhanced oral delivery (e.g., by improving acid resistance and/or resistance to bile acids), to enhance the immunomodulatory and/or therapeutic effect of the EVs they produce (e.g., either alone or in combination with another therapeutic agent), to enhance immune activation by the EVs they produce and/or to improve bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times). The engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.


In some embodiments of the methods provided herein, the bacterium is modified by directed evolution. In some embodiments, the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition. In some embodiments, the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium. In some embodiments, the method further comprises mutagenizing the bacteria (e.g., by exposure to chemical mutagens and/or UV radiation) followed by an assay to detect bacteria having the desired phenotype (e.g., an in vivo assay, an ex vivo assay, or an in vitro assay).


In some embodiments, the bacterium provided herein are modified by exposure to a stress-inducing environment (e.g., an environment that induces envelope stress). In some embodiments, growth under such growth conditions increase production of EVs by the bacterium. For example, in some embodiments, the bacterium is grown in the presence of subinhibitory concentrations of an antibiotic described herein (e.g., 0.1-1 μg/mL chloramphenicol, or 0.1-0.3 μg/mL gentamicin). In some embodiments, host antimicrobial peptides (e.g., lysozyme, defensins, and Reg proteins) are used in place of or in combination with antibiotics. In some embodiments, bacterially-produced antimicrobial peptides (e.g., bacteriocins and microcins) are used. In some embodiments, the stress is temperature stress (e.g., growth at 37-50° C.). In some embodiments, the stress is carbon limitation stress (e.g., growth in a media comprising limited carbon sources, such as media with carbon source restricted below 1% (w/v)). In some embodiments, the stress is salt stress (e.g., growth in a medium containing 0.5M NaCl). In some embodiments, the stress is UV stress (e.g., growth under a UV lamp, either throughout the entire cultivation period or only during a portion of the cultivation period). In some embodiments, the stress is reactive oxygen stress (e.g., growth in media containing subinhibitory concentrations of hydrogen peroxide, such as 250-1,000 μM hydrogen peroxide). In some embodiments, a combination of the stresses disclosed herein are applied to the bacterium.


EXAMPLES
Example 1: Preparation and Purification of EVs from Bacteria

Extracellular vesicles (EVs) are prepared from bacterial cultures using methods known to those skilled in the art (S. Bin Park, et al. PLOS ONE. 6(3): e17629 (2011)).


For example, bacterial cultures are centrifuged at 11,000×g for 20-40 min at 4° C. to pellet bacteria. Culture supernatants are then passed through a 0.22 μm filter to exclude intact bacterial cells. Filtered supernatants are concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. Briefly, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate is added to filtered supernatant slowly, while stirring at 4° C. Precipitations are incubated at 4° C. for 8-48 hours and then centrifuged at 11,000×g for 20-40 min at 4° C. The pellets contain bacterial EVs and other debris. Briefly, using ultracentrifugation, filtered supernatants are centrifuged at 100,000-200,000×g for 1-16 hours at 4° C. The pellet of this centrifugation contains bacterial EVs and other debris. Briefly, using a filtration technique, using an Amicon Ultra spin filter or by tangential flow filtration, supernatants are filtered so as to retain species of molecular weight>50 or 100 kDa.


Alternatively, EVs are obtained from bacterial cultures continuously during growth, or at selected time points during growth, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen) according to manufacturer's instructions. The ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., EVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 um and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.


EVs obtained by methods described above may be further purified by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24 hours at 4° C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. If filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24 hours at 4° C.


To confirm sterility and isolation of the EV preparations, EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.


Alternatively, for preparation of EVs used for in vivo injections, purified EVs are processed as described previously (G. Norheim, et al. PLOS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 μg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).


To make samples compatible with further testing (e.g. to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation (200,000×g, ≥3 hours, 4° C.) and resuspension.


Example 2: Labeling Bacterial EVs

In order to track their biodistribution in vivo and to quantify and localize them in vitro in various preparations and in assays conducted with mammalian cells, EVs are labeled as previously described (N. Kesty, et al. EMBO Journal. 23: 4538-4549 (2004)).


For example, purified EVs are incubated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich, USA), Cy7, or any other fluorochrome suitable for flow cytometry, 1:1 for 1 hour at 25° C. The incubation step may be extended overnight at 4° ° C. To remove extra fluorochrome, EVs are then either (1) pelleted by centrifugation at 200,000×g for 3 hr-overnight, washed, and resuspended in PBS or another appropriate buffer for downstream applications; or (2) buffer exchanged into PBS or another appropriate buffer for downstream applications by dialysis or by filtration (e.g., using an Amicon Ultra column).


Alternatively, EVs are obtained from bacteria cultured in medium containing 0.8 mM 3-azido-D-alanine or HADA. EVs are resuspended or buffer exchanged into PBS and a portion is further labeled with 10 uM Dibenzo-aza-cyclooctyne (DIBAC)-fluorescent dye in 1% BSA/PBS (dyes include Cy5, TAMRA, Rhodamine-green, and Cy7) if grown with 3-azido-D-alanine. Unincorporated dye is removed as described above, by (1) ultracentrifugation, washing, and resuspension; or (2) buffer exchange by dialysis or filtration.


Labeled EVs may also be generated from bacteria expressing green-fluorescent protein (GFP), or any other fluorescent protein. For Gram negative bacteria, periplasmic targeting sequences are appended to the fluorescent proteins, so that they are appropriately localized to be internalized by EVs as they form.


Quantum dots may be used to label EVs for non-invasive in vivo imaging studies (K. Kikushima, et al. Scientific Reports. 3(1913) (2013)). Quantum dots are conjugated to an antibody confirmed to be present in the EV membrane. Isolated EVs are incubated with quantum dot conjugates, and extra conjugates are removed as described above, by (1) ultracentrifugation, washing, and resuspension; or (2) buffer exchange by dialysis or filtration.


Fluorescently labeled EVs are detected in in vitro and ex vivo samples by confocal microscopy, nanoparticle tracking analysis, and/or flow cytometry. Additionally, fluorescently labeled EVs are detected in whole animals and/or dissected organs and tissues using an instrument such as the IVIS spectrum CT (Perkin Elmer), as in H-I. Choi, et al. Experimental & Molecular Medicine. 49: e330 (2017).


Additionally, EVs may be radiolabeled as previously described (Z. Varga et al., Cancer Biother Radiopharm. 2016 June; 31(5):168-73).


For example, purified EVs are radiolabeled with the 99mTc-tricarbonyl complex [99mTc(CO)3(H2O)3]+ using a commercial kit (Isolink®; Mallinckrodt Medical B.V.), according to the manufacturer's instructions.


Example 3: Transmission Electron Microscopy to Visualize Bacterial Production of EVs and Purified Bacterial EVs

Transmission electron microscopy (TEM) is used to visualize bacteria as they produce EVs or purified bacterial EVs (S. Bin Park, et al. PLOS ONE. 6(3): e17629 (2011). EVs are prepared from bacteria batch culture as described in Example 1. EVs are mounted onto 300- or 400-mesh-size carbon-coated copper grids (Electron Microscopy Sciences, USA) for 2 min and washed with deionized water. EVs are negatively stained using 2% (w/v) uranyl acetate for 20 sec-1 min. Copper grids are washed with sterile water and dried. Images are acquired using a transmission electron microscope with 100-120 kV acceleration voltage. Stained EVs appear between 20-250 nm in diameter and are electron dense. 10-50 fields on each grid are screened.


Example 4: Profiling EV Composition and Content

EVs may be characterized by any one of various methods including, but not limited to, NanoSight characterization, SDS-PAGE gel electrophoresis, Western blot, ELISA, liquid chromatography-mass spectrometry and mass spectrometry, dynamic light scattering, lipid levels, total protein, lipid to protein ratios, nucleic acid analysis and zeta potential.


NanoSight Characterization of EVs


Nanoparticle tracking analysis (NTA) is used to characterize the size distribution of purified bacterial EVs. Purified EV preps are run on a NanoSight machine (Malvern Instruments) to assess EV size and concentration.


SDS-PAGE Gel Electrophoresis


To identify the protein components of purified EVs (Example 1), samples are run on a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific) using standard techniques. Samples are boiled in 1×SDS sample buffer for 10 min, cooled to 4° C., and then centrifuged at 16,000×g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (e.g., Silver staining, Coomassie Blue, Gel Code Blue) for visualization of bands.


Western Blot Analysis


To identify and quantify specific protein components of purified EVs, EV proteins are separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et al., Sci. Rep. 6, 36338 (2016) and are quantified via ELISA.


Liquid Chromatography-Mass Spectrometry (LC-MS MS) and Mass Spectrometry (MS)


Proteins present in EVs are identified and quantified by Mass Spectrometry techniques. Additionally, metabolic content is ascertained using liquid chromatography techniques combined with mass spectrometry. A variety of techniques exist to determine metabolomic content of various samples and are known to one skilled in the art involving solvent extraction, chromatographic separation and a variety of ionization techniques coupled to mass determination (Roberts et al 2012 Targeted Metabolomics. Curr Protoc Mol Biol. 30: 1-24; Dettmer et al 2007, Mass spectrometry-based metabolomics. Mass Spectrom Rev. 26(1):51-78). As a non-limiting example, a LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies). Media samples or other complex metabolic mixtures (˜10 μL) are extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). Standards may be adjusted or modified depending on the metabolites of interest. The samples are centrifuged (10 min, 9,000 g, 4° C.), and the supernatants (10 μL) are submitted to LCMS by injecting the solution onto the HILIC column (150×2.1 mm, 3 μm particle size). The column is eluted by flowing a 5% mobile phase [10 mM ammonium formate, 0.1% formic acid in water] for 1 min at a rate of 250 μL/min followed by a linear gradient over 10 min to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid]. The ion spray voltage is set to 4.5 kV and the source temperature is 450° C.


The data are analyzed using commercially available software like Multiquant 1.2 from AB SCIEX for mass spectrum peak integration. Peaks of interest should be manually curated and compared to standards to confirm the identity of the peak. Quantitation with appropriate standards is performed to determine the number of metabolites present in the initial media, after bacterial conditioning and after tumor cell growth.


Dynamic Light Scattering (DLS)


DLS measurements, including the distribution of particles of different sizes in different EV preps are taken using instruments such as the DynaPro NanoStar (Wyatt Technology) and the Zetasizer Nano ZS (Malvern Instruments).


Lipid Levels


Lipid levels are quantified using FM4-64 (Life Technologies), by methods similar to those described by A. J. McBroom et al. J Bacteriol 188:5385-5392. and A. Frias, et al. Microb Ecol. 59:476-486 (2010). Samples are incubated with FM4-64 (3.3 μg/mL in PBS for 10 min at 37° C. in the dark). After excitation at 515 nm, emission at 635 nm is measured using a Spectramax M5 plate reader (Molecular Devices). Absolute concentrations are determined by comparison of unknown samples to standards (such as palmitoyloleoylphosphatidylglycerol (POPG) vesicles) of known concentrations.


Total Protein


Protein levels are quantified by standard assays such as the Bradford and BCA assays. The Bradford assays are run using Quick Start Bradford 1× Dye Reagent (Bio-Rad), according to manufacturer's protocols. BCA assays are run using the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific). Absolute concentrations are determined by comparison to a standard curve generated from BSA of known concentrations.


Lipid:Protein Ratios


Lipid:protein ratios are generated by dividing lipid concentrations by protein concentrations. These provide a measure of the purity of vesicles as compared to free protein in each preparation.


Nucleic Acid Analysis


Nucleic acids are extracted from EVs and quantified using a Qubit fluorometer. Size distribution is assessed using a BioAnalyzer and the material is sequenced.


Zeta Potential


The zeta potential of different preparations are measured using instruments such as the Zetasizer ZS (Malvern Instruments).


Example 5: Manipulating Bacteria Through Stress to Produce Various Amounts of EVs and/or to Vary Content of EVs

Stress, and in particular envelope stress, has been shown to increase production of EVs by some bacterial strains (I. MacDonald, M. Kuehn. J Bacteriol 195(13): doi: 10/1128/JB.02267-12). In order to vary production of EVs by bacteria, bacteria are stressed using various methods.


Bacteria may be subjected to single stressors or stressors in combination. The effects of different stressors on different bacteria is determined empirically by varying the stress condition and determining the IC50 value (the conditions required to inhibit cell growth by 50%). EV purification, quantification, and characterization occurs as detailed in Examples 1-4. EV production is quantified (1) in complex samples of bacteria and EVs by nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM); or (2) following EV purification by NTA, lipid quantification, or protein quantification. EV content is assessed following purification by methods described above.


Antibiotic Stress


Bacteria are cultivated under standard growth conditions with the addition of subinhibitory concentrations of antibiotics. This may include 0.1-1 μg/mL chloramphenicol, or 0.1-0.3 μg/mL gentamicin, or similar concentrations of other antibiotics (e.g., ampicillin, polymyxin B). Host antimicrobial products such as lysozyme, defensins, and Reg proteins may be used in place of antibiotics. Bacterially-produced antimicrobial peptides, including bacteriocins and microcins may also be used.


Temperature Stress


Bacteria are cultivated under standard growth conditions, but at higher or lower temperatures than are typical for their growth. Alternatively, bacteria are grown under standard conditions, and then subjected to cold shock or heat shock by incubation for a short period of time at low or high temperatures respectively. For example, bacteria grown at 37° C. are incubated for 1 hour at 4° C.-18° C. for cold shock or 42° C.-50° C. for heat shock.


Starvation and Nutrient Limitation


To induce nutritional stress, bacteria are cultivated under conditions where one or more nutrients are limited. Bacteria may be subjected to nutritional stress throughout growth or shifted from a rich medium to a poor medium. Some examples of media components that are limited are carbon, nitrogen, iron, and sulfur. An example medium is M9 minimal medium (Sigma-Aldrich), which contains low glucose as the sole carbon source. Particularly for Prevotella spp., iron availability is varied by altering the concentration of hemin in media and/or by varying the type of porphyrin or other iron carrier present in the media, as cells grown in low hemin conditions were found to produce greater numbers of EVs (S. Stubbs et al. Letters in Applied Microbiology. 29:31-36 (1999). Media components are also manipulated by the addition of chelators such as EDTA and deferoxamine.


Saturation


Bacteria are grown to saturation and incubated past the saturation point for various periods of time. Alternatively, conditioned media is used to mimic saturating environments during exponential growth. Conditioned media is prepared by removing intact cells from saturated cultures by centrifugation and filtration, as described in Example 1, and conditioned media may be further treated to concentrate or remove specific components.


Salt Stress


Bacteria are cultivated in or exposed for brief periods to medium containing NaCl, bile salts, or other salts.


UV Stress


UV stress is achieved by cultivating bacteria under a UV lamp or by exposing bacteria to UV using an instrument such as a Stratalinker (Agilent). UV may be administered throughout the entire cultivation period, in short bursts, or for a single defined period following growth.


Reactive Oxygen Stress


Bacteria are cultivated in the presence of subinhibitory concentrations of hydrogen peroxide (250-1,000 μM) to induce stress in the form of reactive oxygen species. Anaerobic bacteria are cultivated in or exposed to concentrations of oxygen that are toxic to them.


Detergent Stress


Bacteria are cultivated in or exposed to detergent, such as sodium dodecyl sulfate (SDS) or deoxycholate.


pH Stress


Bacteria are cultivated in or exposed for limited times to media of different pH.


Example 6: Preparation of EV-Free Bacteria

Bacterial samples containing minimal amounts of EVs are prepared. EV production is quantified (1) in complex samples of bacteria and extracellular components by NTA or TEM; or (2) following EV purification from bacterial samples, by NTA, lipid quantification, or protein quantification.

    • a. Centrifugation and washing: Bacterial cultures are centrifuged at 11,000×g to separate intact cells from supernatant (including free proteins and vesicles). The pellet is washed with buffer, such as PBS, and stored in a stable way (e.g., mixed with glycerol, flash frozen, and stored at)−80° ° C.
    • b. ATF: Bacteria and EVs are separated by connection of a bioreactor to an ATF system. EV-free bacteria are retained within the bioreactor, and may be further separated from residual EVs by centrifugation and washing, as described above.
    • c. Bacteria are grown under conditions that are found to limit production of EVs. Conditions that may be varied include those listed for Example 5.


Example 7: In Vitro Screening of EVs for Enhanced Activation of Dendritic Cells

The ability of Vibrio cholerae EVs to activate dendritic cells indirectly through epithelial cells is one nonlimiting mechanism by which they stimulate an immune response in mammalian hosts (D. Chatterjee, K. Chadhuri. J Biol Chem. 288(6):4299-309. (2013)). As this EV activity is likely shared with other bacteria that stimulate pro-inflammatory cascades in vivo, in vitro methods to assay DC activation by bacterial EVs are disclosed herein. Briefly, PBMCs are isolated from heparinized venous blood from CMs by gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) or from mouse spleens or bone marrow using the magnetic bead-based Human Blood Dendritic cell isolation kit (Miltenyi Biotech, Cambridge, MA). Using anti-human CD14 mAb, the monocytes are purified by Moflo and cultured in cRPMI at a cell density of 5e5 cells/ml in a 96-well plate (Costar Corp) for 7 days at 37° C. For maturation of dendritic cells, the culture is stimulated with 0.2 ng/ml IL-4 and 1000 U/ml GM-CSF at 37° ° C. for one week. Alternatively, maturation is achieved through incubation with recombinant GM-CSF alone for a week. Mouse DCs can be harvested directly from spleens using bead enrichment or differentiated from haematopheotic stem cells. Briefly, bone marrow is obtained from the femurs of mice. Cells are recovered and red blood cells lysed. Stem cells are cultured in cell culture medium together with 20 ng/ml mouse GMCSF for 4 days. Additional medium containing 20 ng/ml mouse GM-CSF is added. On day 6 the medium and non-adherent cells are removed and replaced with fresh cell culture medium containing 20 ng/ml GMCSF. A final addition of cell culture medium with 20 ng/ml GM-CSF is added on day 7. On day 10 non-adherent cells are harvested and seeded into cell culture plates overnight and stimulated as required. Dendritic cells are then treated with 25-75 μg/mL EVs for 24 hours with antibiotics. EV compositions tested may include a EVs from a single bacterial species or strain. EV compositions tested may also include a mixture of EVs from bacterial genera, species within a genus, or strains within a species. PBS and EVs from Lactobacillus are included as negative controls and LPS, anti-CD40 antibodies, and EVs from Bifidobacterium spp. are used as positive controls. Following incubation, DCs are stained with anti CD11b, CD11c, CD103, CD8a, CD40, CD80, CD83, CD86, MHCI and MHCII, and analyzed by flow cytometry. DCs that are significantly increased in CD40, CD80, CD83, and CD86 as compared to negative controls are considered to be activated by the associated bacterial EV composition. These experiments are repeated three times at minimum.


To screen for the ability of EV-activated epithelial cells to stimulate DCs, the above protocol is followed with the addition of a 24-hour epithelial cell EV co-culture prior to incubation with DCs. Epithelial cells are washed after incubation with EVs and are then co-cultured with DCs in an absence of EVs for 24 hours before being processed as above. Epithelial cell lines may include Int407, HEL293, HT29, T84 and CACO2.


As an additional measure of DC activation, 100 μl of culture supernatant is removed from wells following 24 hour incubation of DCs with EVs or EV-treated epithelial cells and is analyzed for secreted cytokines, chemokines, and growth factors using the multiplexed Luminex Magpix. Kit (EMD Millipore, Darmstadt, Germany). Briefly, the wells are pre-wet with buffer, and 25 μl of 1× antibody-coated magnetic beads are added and 2×200 μl of wash buffer are performed in every well using the magnet. 50 μl of Incubation buffer, 50 μl of diluent and 50 μl of samples are added and mixed via shaking for 2 hrs at room temperature in the dark. The beads are then washed twice with 200 μl wash buffer. 100 μl of 1× biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 μl washes are then performed with wash buffer. 100 μl of 1×SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 μl washes are performed and 125 μl of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.


Standards allow for careful quantitation of the cytokines including GM-CSF, IFN-g, IFN-a, IFN-B, IL-1a, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL-17A, IL-17F, IL-21, IL-22 IL-23, IL-25, IP-10, KC, MCP-1, MIG, MIP1a, TNFa, and VEGF. These cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host. Other variations on this assay examining specific cell types ability to release cytokines are assessed by acquiring these cells through sorting methods and are recognized by one of ordinary skill in the art. Furthermore, cytokine mRNA is also assessed to address cytokine release in response to a EV composition. These changes in the cells of the host stimulate an immune response similarly to in vivo response in a cancer microenvironment.


This DC stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.


Example 8: In Vitro Screening of EVs for Enhanced Activation of CD8+ T Cell Killing when Incubated with Tumor Cells

In vitro methods for screening for EVs that can activate CD8+ T cell killing of tumor cells are described. Briefly, DCs are isolated from human PBMCs or mouse spleens and incubated with single-strain EVs, mixtures of EVs, and appropriate controls as described in Example 12. In addition, CD8+ T cells are obtained from human PBMCs or mouse spleens using the magnetic bead-based Mouse CD8a+ T Cell Isolation Kit and the magnetic bead-based Human CD8+ T Cell Isolation Kit (both from Miltenyi Biotech, Cambridge, MA). After the 24 hour incubation of DCs with EVs, or DCs with EV-stimulated epithelial cells (detailed in Example 12), EVs are removed from cells with PBS washes, 100 μl of fresh media with antibiotics is added to each well, and 200,000 T cells are added to each experimental well in the 96-well plate. Anti-CD3 antibody is added at a final concentration of 2 μg/ml. Co-cultures are then allowed to incubate at 37° ° C. for 96 hours under normal oxygen conditions.


72 hours into the coculture incubation, 50,000 tumor cells/well are plated per well in new 96-well plates. Mouse tumor cell lines used include B16.F10, SIY+B16.F10, and others. Human tumor cell lines are HLA-matched to donor, and can include PANC-1, UNKPC960/961, UNKC, and HELA cell lines. After completion of the 96 hour co-culture, 100 μl of the CD8+ T cell and DC mixture is transferred to wells containing tumor cells. Plates are incubated for 24 hours at 37° C. under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.


Following this incubation, flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well. Cytotoxic CD8+ T cell phenotype may be characterized by the following methods: a) concentration of supernatant granzyme B, IFNy and TNFa in the culture supernatant as described below, b) CD8+ T cell surface expression of activation markers such as DC69, CD25, CD154, PD-1, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellular cytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells. CD4+ T cell phenotype may also be assessed by intracellular cytokine staining in addition to supernatant cytokine concentration including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.


As an additional measure of CD8+ T cell activation, 100 μl of culture supernatant is removed from wells following the 96 hour incubation of T cells with DCs and is analyzed for secreted cytokines, chemokines, and growth factors using the multiplexed Luminex Magpix. Kit (EMD Millipore, Darmstadt, Germany). Briefly, the wells are pre-wet with buffer, and 25 μl of 1× antibody-coated magnetic beads are added and 2×200 μl of wash buffer are performed in every well using the magnet. 50 μl of Incubation buffer, 50 μl of diluent and 50 μl of samples are added and mixed via shaking for 2 hrs at room temperature in the dark. The beads are then washed twice with 200 μl wash buffer. 100 μl of 1× biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 μl washes are then performed with wash buffer. 100 μl of 1×SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 μl washes are performed and 125 μl of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.


Standards allow for careful quantitation of the cytokines including GM-CSF, IFN-g, IFN-a, IFN-B IL-1a, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-1, MIG, MIP1a, TNFa, and VEGF. These cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host. Other variations on this assay examining specific cell types ability to release cytokines are assessed by acquiring these cells through sorting methods and are recognized by one of ordinary skill in the art. Furthermore, cytokine mRNA is also assessed to address cytokine release in response to an EV composition. These changes in the cells of the host stimulate an immune response similarly to in vivo response in a cancer microenvironment.


This CD8+ T cell stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.


Example 9: In Vitro Screening of EVs for Enhanced Tumor Cell Killing by PBMCs

Methods to screen EVs for the ability to stimulate PBMCs, which in turn activate CD8+ T cells to kill tumor cells are included. PBMCs are isolated from heparinized venous blood from CMs by ficoll-paque gradient centrifugation for mouse or human blood, or with Lympholyte Cell Separation Media (Cedarlane Labs, Ontario, Canada) from mouse blood. PBMCs are incubated with single-strain EVs, mixtures of EVs, and appropriate controls as described in Example 12. In addition, CD8+ T cells are obtained from human PBMCs or mouse spleens as in Example 12. After the 24 hour incubation of PBMCs with EVs, EVs are removed from cells with PBS washes, 100 μl of fresh media with antibiotics is added to each well, and 200,000 T cells are added to each experimental well in the 96-well plate. Anti-CD3 antibody is added at a final concentration of 2 μg/ml. Co-cultures are then allowed to incubate at 37° C. for 96 hours under normal oxygen conditions.


72 hours into the coculture incubation, 50,000 tumor cells/well are plated per well in new 96-well plates. Mouse tumor cell lines used include B16.F10, SIY+B16.F10, and others. Human tumor cell lines are HLA-matched to donor, and can include PANC-1, UNKPC960/961, UNKC, and HELA cell lines. After completion of the 96 hour co-culture, 100 μl of the CD8+T cell and PBMC mixture is transferred to wells containing tumor cells. Plates are incubated for 24 hours at 37° C. under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.


Following this incubation, flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well. Cytotoxic CD8+ T cell phenotype may be characterized by the following methods: a) concentration of supernatant granzyme B, IFNy and TNFa in the culture supernatant as described below, b) CD8+ T cell surface expression of activation markers such as DC69, CD25, CD154, PD-1, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellular cytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells. CD4+ T cell phenotype may also be assessed by intracellular cytokine staining in addition to supernatant cytokine concentration including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.


As an additional measure of CD8+ T cell activation, 100 μl of culture supernatant is removed from wells following the 96 hour incubation of T cells with DCs and is analyzed for secreted cytokines, chemokines, and growth factors using the multiplexed Luminex Magpix. Kit (EMD Millipore, Darmstadt, Germany). Briefly, the wells are pre-wet with buffer, and 25 μl of 1× antibody-coated magnetic beads are added and 2×200 μl of wash buffer are performed in every well using the magnet. 50 μl of Incubation buffer, 50 μl of diluent and 50 μl of samples are added and mixed via shaking for 2 hrs at room temperature in the dark. The beads are then washed twice with 200 μl wash buffer. 100 μl of 1× biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 μl washes are then performed with wash buffer. 100 μl of 1×SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 μl washes are performed and 125 μl of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.


Standards allow for careful quantitation of the cytokines including GM-CSF, IFN-g, IFN-a, IFN-B IL-1a, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-1, MIG, MIP1a, TNFa, and VEGF. These cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host. Other variations on this assay examining specific cell types ability to release cytokines are assessed by acquiring these cells through sorting methods and are recognized by one of ordinary skill in the art. Furthermore, cytokine mRNA is also assessed to address cytokine release in response to an EV composition. These changes in the cells of the host stimulate an immune response similarly to in vivo response in a cancer microenvironment.


This PBMC stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.


Example 10. In Vitro Detection of EVs in Antigen-Presenting Cells

Dendritic cells in the lamina propria constantly sample live bacteria, dead bacteria, and microbial products in the gut lumen by extending their dendrites across the gut epithelium, which is one way that EVs produced by bacteria in the intestinal lumen may directly stimulate dendritic cells. The following methods represent a way to assess the differential uptake of EVs by antigen-presenting cells. Optionally, these methods may be applied to assess immunomodulatory behavior of EVs administered to a patient.


Dendritic cells (DCs) are isolated from human or mouse bone marrow, blood, or spleens according to standard methods or kit protocols (e.g., Inaba K, Swiggard W J, Steinman RM, Romani N, Schuler G, 2001. Isolation of dendritic cells. Current Protocols in Immunology. Chapter 3: Unit3.7) and as discussed in Example 12.


To evaluate EV entrance into and/or presence in DCs, 250,000 DCs are seeded on a round cover slip in complete RPMI-1640 medium and are then incubated with EVs from single bacterial strains or combinations EVs at a multiplicity of infection (MOI) between 1:1 and 1:10. Purified EVs have been labeled with fluorochromes or fluorescent proteins as described in Example 2. After 1 hour of incubation, the cells are washed twice with ice-cold PBS, detached from the plate using trypsin. Cells are either allowed to remain intact or are lysed. Samples are then processed for flow cytometry. Total internalized EVs are quantified from lysed samples, and percentage of cells that uptake EVs is measured by counting fluorescent cells. The methods described above may also be performed in substantially the same manner using macrophages or epithelial cell lines (obtained from the ATCC) in place of DCs.


Example 11: In Vitro Screening of EVs with an Enhanced Ability to Activate NK Cell Killing when Incubated with Target Cells

To demonstrate the ability of the selected EV compositions to elicit potent NK cell cytotoxicity to tumor cells when incubated with the tumor cells, the following in vitro assay is used. Briefly, mononuclear cells from heparinized blood are obtained from healthy human donors. Optionally, an expansion step to increase the numbers of NK cells is performed as previously described (e.g. see Somanschi et al 2011 J Vis Exp.). They are adjusted to a concentration of 1e6 cells/ml in RPMI-1640 medium containing 5% human serum. The PMNC cells are then labeled with appropriate antibodies and NK cells are isolated through FACS as CD3−/CD56+ cells and are ready for the subsequent cytotoxicity assay. Alternatively, NK cells are isolated using the autoMACs instrument and NK cell isolation kit following manufacturer's instructions (Miltenyl Biotec).


NK cells are counted, and plated in a 96 well format with 5000 cells per well, and incubated with single-strain EVs, EVs from mixtures of bacterial strains, and appropriate controls as described in Example 12. As an additional negative control, this assay is run with EVs from Fusobacterium nucleatum. F. nucleatum is known to be inhibitory to NK cell activity (see e.g. Gur et al 2005 Immunity 42:1-12). After 5-24 hours incubation of NK cells with EVs, EVs are removed from cells with PBS washes, NK cells are resuspended in 10 fresh media with antibiotics, and are added to 96-well plates containing 50,000 target tumor cells/well. Mouse tumor cell lines used include B16.F10, SIY+B16.F10, and others. Human tumor cell lines are HLA-matched to donor, and can include PANC-1, UNKPC960/961, UNKC, and HELA cell lines. Plates are incubated for 24 hours at 37° C. under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.


Following this incubation, flow cytometry is used to measure tumor cell death. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well.


This NK cell stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.


Example 12: Using In Vitro Immune Activation Assays to Predict In Vivo Cancer Immunotherapy Efficacy of EV Compositions

In vitro immune activation assays identify EVs that are able to stimulate dendritic cells, which in turn activate CD8+ T cell killing. Work by A. Sivan, et al, Science 350(6264): 1084-1089 (2015) has suggested that enhanced killing of tumor cells by CD8+ T cells in response to oral ingestion of Bifidobacterium spp. is an effective cancer immunotherapy in mice. Therefore, the in vitro assays described above are used as a predictive, fast screen of a large number of candidate EVs for potential immunotherapy activity. EVs that display enhanced stimulation of dendritic cells, enhanced stimulation of CD8+ T cell killing, enhanced stimulation of PBMC killing, and/or enhanced stimulation of NK cell killing, are preferentially chosen for in vivo cancer immunotherapy efficacy studies.


Example 13: Determining the Biodistribution of EVs when Delivered Orally to Mice

Wild-type mice (e.g., C57BL/6 or BALB/c) are orally inoculated with the EV composition of interest to determine the in vivo biodistibution profile of purified EVs (Example 1). EVs are labeled as in Example 2 to aide in downstream analyses.


Mice can receive a single dose of the EV (25-100 μg) or several doses over a defined time course (25-100 μg). Mice are housed under specific pathogen-free conditions following approved protocols. Alternatively, mice may be bred and maintained under sterile, Germ-free conditions. Blood and stool samples can be taken at appropriate time points.


The mice are humanely sacrificed at various time points (i.e., hours to days) post inoculation with the EV compositions and a full necropsy under sterile conditions is performed. Following standard protocols, lymph nodes, adrenal glands, liver, colon, small intestine, cecum, stomach, spleen, kidneys, bladder, pancreas, heart, skin, lungs, brain, and other tissue of interest are harvested and are used directly or snap frozen for further testing. The tissue samples are dissected and homogenized to prepare single-cell suspensions following standard protocols known to one skilled in the art. The number of EVs present in the sample is then quantified through flow cytometry (Example 17). Quantification may also proceed with use of fluorescence microscopy after appropriate processing of whole mouse tissue (Vankelecom H., Fixation and paraffin-embedding of mouse tissues for GFP visualization, Cold Spring Harb. Protoc., 2009). Alternatively, the animals may be analyzed using live-imaging according to the EV labeling technique.


Example 14: Administering EV Compositions with Enhanced Immune Activation In Vitro to Treat Syngeneic Mouse Tumor Models

A mouse model of cancer is generated by subcutaneously injecting a tumor cell line or patient derived tumor sample and allowing it to engraft into C57BL/6, female mice at ages 6-8 weeks old. The methods provided herein are replicated using several tumor cell lines including: B16-F10 or B16-F10-SIY cells as an orthotopic model of melanoma, Panc02 cells as an orthotopic model of pancreatic cancer, injected at a concentration of 1×106 cells into the right flank (Maletzki et al 2008. Gut 57:483-491), LLC1 cells as an orthotopic model of lung cancer, CT-26 as an orthotopic model of colorectal cancer, and RM-1 as an orthotopic model of prostate cancer. As an example, methods for the B16-F10 model are provided in depth herein.


A syngeneic mouse model of spontaneous melanoma with a very high metastatic frequency is used to test the ability of bacteria to reduce tumor growth and the spread of metastases. The EVs chosen for this assay are compositions that display enhanced activation of immune cell subsets and stimulate enhanced killing of tumor cells in vitro (Examples 12-16). The mouse melanoma cell line B16-F10 is obtained from ATCC. The cells are cultured in vitro as a monolayer in RPMI medium, supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37° C. in an atmosphere of 5% CO2 in air. The exponentially growing tumor cells are harvested by trypsinization, washed three times with cold 1×PBS, and a suspension of 5E6 cells/ml is prepared for administration. Female C57BL/6 mice are used for this experiment. The mice are 6-8 weeks old and weigh approximately 16-20 g. For tumor development, each mouse is injected SC into the flank with 100 μl of the B16-F10 cell suspension. The mice are anesthetized by ketamine and xylazine prior to the cell transplantation. The animals used in the experiment may be started on an antibiotic treatment via instillation of a cocktail of kanamycin (0.4 mg/ml), gentamicin, (0.035 mg/ml), colistin (850 U/ml), metronidazole (0.215 mg/ml) and vancomycin (0.045 mg/ml) in the drinking water from day 2 to 5 and an intraperitoneal injection of clindamycin (10 mg/kg) on day 7 after tumor injection.


The size of the primary flank tumor is measured with a caliper every 2-3 days and the tumor volume is calculated using the following formula: tumor volume=the tumor width2×tumor length×0.5. After the primary tumor reaches approximately 100 mm3, the animals are sorted into several groups based on their body weight. The mice are then randomly taken from each group and assigned to a treatment group. EV compositions are prepared as described in Example 1. The mice are orally inoculated by gavage with either 25-100 μg EV to be tested, 25-100 μg EV from Lactobacillus (negative control), PBS, or 25-100 μg EV from Bifidobacterium spp. (positive control). Mice are orally gavaged with the same amount of EVs daily, weekly, bi-weekly, monthly, bi-monthly, or on any other dosing schedule throughout the treatment period. Mice are IV injected in the tail vein or directly injected into the tumor. Mice can be injected with 10 ng-1 ug of EVs, bacteria and EVs or inactivated bacteria and EVs. Mice can be injected weekly or once a month. Mice may also receive combinations of purified EVs and live bacteria to maximize tumor-killing potential. All mice are housed under specific pathogen-free conditions following approved protocols. Tumor size, mouse weight, and body temperature are monitored every 3-4 days and the mice are humanely sacrificed 6 weeks after the B16-F10 mouse melanoma cell injection or when the volume of the primary tumor reaches 1000 mm3. Blood draws are taken weekly and a full necropsy under sterile conditions is performed at the termination of the protocol.


Cancer cells can be easily visualized in the mouse B16-F10 melanoma model due to their melanin production. Following standard protocols, tissue samples from lymph nodes and organs from the neck and chest region are collected and the presence of micro- and macro-metastases is analyzed using the following classification rule. An organ is classified as positive for metastasis if at least two micro-metastatic and one macro-metastatic lesion per lymph node or organ are found. Micro-metastases are detected by staining the paraffin-embedded lymphoid tissue sections with hematoxylin-eosin following standard protocols known to one skilled in the art. The total number of metastases is correlated to the volume of the primary tumor and it is found that the tumor volume correlates significantly with tumor growth time and the number of macro- and micro-metastases in lymph nodes and visceral organs and also with the sum of all observed metastases. Twenty-five different metastatic sites are identified as previously described (Bobek V., et al., Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma, Clin. Exp. Metastasis, 2004; 21(8):705-8).


The tumor tissue samples are further analyzed for tumor infiltrating lymphocytes. The CD8+ cytotoxic T cells can be isolated by FACS (see Example 17) and can then be further analyzed using customized p/MHC class I microarrays to reveal their antigen specificity (see e.g. Deviren G., et al., Detection of antigen-specific T cells on p/MHC microarrays, J. Mol. Recognit., 2007 January-February; 20(1):32-8). CD4+ T cells can be analyzed using customized p/MHC class II microarrays.


The same experiment is also performed with a mouse model of multiple pulmonary melanoma metastases. The mouse melanoma cell line B16-BL6 is obtained from ATCC and the cells are cultured in vitro as described above. Female C57BL/6 mice are used for this experiment. The mice are 6-8 weeks old and weigh approximately 16-20 g. For tumor development, each mouse is injected into the tail vein with 100 μl of a 2E6 cells/ml suspension of B16-BL6 cells. The tumor cells that engraft upon IV injection end up in the lungs.


The mice are humanely killed after 9 days. The lungs are weighed and analyzed for the presence of pulmonary nodules on the lung surface. The extracted lungs are bleached with Fekete's solution, which does not bleach the tumor nodules because of the melanin in the B16 cells though a small fraction of the nodules is amelanotic (i.e. white). The number of tumor nodules is carefully counted to determine the tumor burden in the mice. Typically, 200-250 pulmonary nodules are found on the lungs of the control group mice (i.e. PBS gavage).


The percentage tumor burden is calculated for the three treatment groups. This measure is defined as the mean number of pulmonary nodules on the lung surfaces of mice that belong to a treatment group divided by the mean number of pulmonary nodules on the lung surfaces of the control group mice.


Determining Metabolic Content with H-NMRI


Biological triplicates of media and spent media samples after bacterial conditioning and after growth of the tumor are deproteinized using Sartorius Centrisart I filters (cutoff 10 kDa). Before use, the filter is washed twice by centrifugation of water to remove glycerol and a small volume (20 μl) of 20.2 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP, sodium salt) in D2O is added to 700 μl of the ultrafiltrate, providing a chemical shift reference (0.00 ppm) and a deuterium lock signal. 650 μl of the sample is placed in a 5 mm NMR tube. Single pulse 1H-NMR spectra (500 MHZ) are obtained on a Bruker DMX-500 spectrometer or comparable instrument as described previously (by Engelke et al. 2006 NMR spectroscopic studies on the late onset form of 3-methylutaconic aciduria type I and other defects in leucine metabolism. NMR Biomed. 19: 271-278). Phase and baseline are corrected manually. All spectra are scaled to TSP and metabolite signals are fitted semi-automatically with a Lorentzian line shape. Metabolite concentrations in the spent media are calculated relative to the known concentration in the standard medium and correspondingly expressed in units of mM. The concentration of a particular metabolite was calculated by the area of the corresponding peak to the area of the valine doublet at 1.04 ppm or an appropriate standard.


Determining Metabolic Content with LCMS


Metabolic content of a sample is ascertained using liquid chromatography techniques combined with mass spectrometry. A variety of techniques exist to determine metabolomic content of various samples and are known to one skilled in the art involving solvent extraction, chromatographic separation and a variety of ionization techniques coupled to mass determination (Roberts et al 2012 Targeted Metabolomics. Curr Protoc Mol Biol. 30: 1-24; Dettmer et al 2007, Mass spectrometry-based metabolomics. Mass Spectrom Rev. 26(1):51-78). As a non-limiting example, a LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies). Media samples or other complex metabolic mixtures (˜10 μL) are extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). Standards may be adjusted or modified depending on the metabolites of interest. The samples are centrifuged (10 min, 9,000 g, 4° C.), and the supernatants (10 μL) are submitted to LCMS by injecting the solution onto the HILIC column (150×2.1 mm, 3 μm particle size). The column is eluted by flowing a 5% mobile phase [10 mM ammonium formate, 0.1% formic acid in water] for 1 min at a rate of 250 μL/min followed by a linear gradient over 10 min to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid]. The ion spray voltage is set to 4.5 kV and the source temperature is 450° C.


The data are analyzed using commercially available software such as Multiquant 1.2 from AB SCIEX for mass spectrum peak integration. Peaks of interest are manually curated and compared to standards to confirm the identity of the peak. Quantitation with appropriate standards is performed to determine the amount of metabolites present in the initial media, after bacterial conditioning and after tumor cell growth.


The tumor biopsies and blood samples are submitted for metabolic analysis via LCMS techniques described herein. Differential levels of amino acids, sugars, lactate, among other metabolites, between test groups demonstrate the ability of the microbial composition to disrupt the tumor metabolic state.


RNA Seq to Determine Mechanism of Action


Dendritic cells are purified from tumors, Peyers patches, and mesenteric lymph nodes as described in Example 12. RNAseq analysis is carried out and analyzed according to standard techniques known to one skilled in the art (Z. Hou. Scientific Reports. 5(9570):doi: 10.1038/srep09570 (2015)). In the analysis, specific attention is placed on innate inflammatory pathway genes including TLRs, CLRs, NLRs, and STING, cytokines, chemokines, antigen processing and presentation pathways, cross presentation, and T cell co-stimulation.


Example 15: Administering EVs with Enhanced Immune Activation In Vitro to Treat Syngeneic Mouse Tumor Models in Combination with PD-1 or PD-L1 Inhibition

To determine the efficacy of EVs in syngeneic tumor mouse models, colorectal cancer (CT-26) was used. Briefly, CT-26 (CAT #CRL-2638) tumor cells were cultured in vitro as a monolayer in RPMI-1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The exponentially-growing cells were harvested and counted prior to tumor inoculation. 6-8 week old female BALB/c mice were used for this experiment. For tumor development, each mouse was injected subcutaneously in one or both rear flanks with 5×105 CT-26 tumor cells in 0.1 ml of 1×PBS. Some mice may receive antibiotic pre-treatment. Tumor size and mouse weight were monitored at least thrice weekly on nonconsecutive days.


EVs were tested for their efficacy in the mouse tumor model, either alone or in combination with whole bacterial cells and with or without anti-PD-1 or anti-PD-L1. EVs, bacterial cells, and/or anti-PD-1 or anti-PD-L1 were administered at varied time points and at varied doses. For example, on day 10 after tumor injection, or after the tumor volume reaches 100 mm3, the mice were treated with EVs alone or in combination with anti-PD-1 or anti-PD-L1. Dosing amount was administered according to table 3. The Blautia massiliensis group showed tumor growth inhibition comparable to that seen in the anti-PD-1 group (FIGS. 1 and 2).









TABLE 3







Dosing amount.









Group
Treatment
Dose, route, schedule





1 (n = 10)
IV Vehicle (PBS)
N/A, IV, Q3Dx4


2 (n = 10)
Anti-PD-1
200 ug, IP, Q4Dx3


3 (n = 10)
EV (IV) Blautia massiliensis
5 ug, IV, Q3Dx4









For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mgs of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration. For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route injection. Some groups of mice are also injected with effective doses of checkpoint inhibitor. For example, mice receive 100 μg anti-PD-L1 mAB (clone 10f.9 g2, BioXCell) or another anti-PD-1 or anti-PD-L1 mAB in 100 μl PBS, and some mice receive vehicle and/or other appropriate control (e.g. control antibody). Mice are injected with mABs 3, 6, and 9 days after the initial injection. To assess whether checkpoint inhibition and EV immunotherapy have an additive, anti-tumor effect, control mice receiving anti-PD-1 or anti-PD-L1 mABs are included to the standard control panel. Primary (tumor size) and secondary (tumor infiltrating lymphocytes and cytokine analysis) endpoints are assessed, and some groups of mice are rechallenged with a subsequent tumor cell inoculation to assess the effect of treatment on memory response.


Example 16: EVs in a Mouse Model of Experimental Autoimmune Encephalomyelitis (EAE)

EAE is a well-studied animal model of multiple sclerosis, as reviewed by Constantinescu et al. (Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 2011 October; 164(4): 1079-1106). It can be induced in a variety of mouse and rat strains using different myelin-associated peptides, by the adoptive transfer of activated encephalitogenic T cells, or the use of TCR transgenic mice susceptible to EAE, as discussed in Mangalam et al. (Two discreet subsets of CD8+ T cells modulate PLP91-110 induced experimental autoimmune encephalomyelitis in HLA-DR3 transgenic mice. J Autoimmun. 2012 June; 38(4): 344-353).


EVs are tested for their efficacy in the rodent model of EAE, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, female 6-8 week old C57Bl/6 mice are obtained from Taconic (Germantown, NY). Groups of mice are administered two subcutaneous (s.c.) injections at two sites on the back (upper and lower) of 0.1 ml myelin oligodentrocyte glycoprotein 35-55 (MOG35-55; 100 ug per injection; 200 ug per mouse (total 0.2 ml per mouse)), emulsified in Complete Freund's Adjuvant (CFA; 2-5 mg killed Mycobacterium tuberculosis H37Ra/ml emulsion). Approximately 1-2 hours after the above, mice are intraperitoneally (i.p.) injected with 200 ng Pertussis toxin (PTx) in 0.1 ml PBS (2 ug/ml). An additional IP injection of PTx is administered on day 2. Alternatively, an appropriate amount of an alternative myelin peptide (e.g. proteolipid protein (PLP)) is used to induce EAE. Some animals serve as naïve controls. EAE severity is assessed and a disability score is assigned daily beginning on day 4 according to methods known in the art (Mangalam et al. 2012).


Treatment with EVs is initiated at some point, either around the time of immunization or following EAE immunization. For example, EVs may be administered at the same time as immunization (day 1), or they may be administered upon the first signs of disability (e.g. limp tail), or during severe EAE. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.) injection, or nasal route administration.


Some groups of mice may be treated with additional anti-inflammatory agent(s) or EAE therapeutic(s) (e.g. anti-CD154, blockade of members of the TNF family, Vitamin D, or other treatment), and/or an appropriate control (e.g. vehicle or control antibody) at various time points and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics.


At various timepoints, mice are sacrificed and sites of inflammation (e.g. brain and spinal cord), lymph nodes, or other tissues may be removed for ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. For example, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+ central nervous system (CNS)-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be rechallenged with a disease trigger (e.g. activated encephalitogenic T cells or re-injection of EAE-inducing peptides). Mice are analyzed for susceptibility to disease and EAE severity following rechallenge.


Example 17: EVs in a Mouse Model of Collagen-Induced Arthritis (CIA)

Collagen-induced arthritis (CIA) is an animal model commonly used to study rheumatoid arthritis (RA), as described by Caplazi et al. (Mouse models of rheumatoid arthritis. Veterinary Pathology. Sep. 1, 2015. 52(5): 819-826) (see also Brand et al. Collagen-induced arthritis. Nature Protocols. 2007. 2: 1269-1275; Pietrosimone et al. Collagen-induced arthritis: a model for murine autoimmune arthritis. Bio Protoc. 2015 Oct. 20; 5(20): e1626).


Among other versions of the CIA rodent model, one model involves immunizing HLA-DQ8 Tg mice with chick type II collagen as described by Taneja et al. (J. Immunology. 2007. 56: 69-78; see also Taneja et al. J. Immunology 2008. 181: 2869-2877; and Taneja et al. Arthritis Rheum., 2007. 56: 69-78). Purification of chick CII has been described by Taneja et al. (Arthritis Rheum., 2007. 56: 69-78). Mice are monitored for CIA disease onset and progression following immunization, and severity of disease is evaluated and “graded” as described by Wooley, J. Exp. Med. 1981. 154: 688-700.


Mice are immunized for CIA induction and separated into various treatment groups. EVs are tested for their efficacy in CIA, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments.


Treatment with EVs is initiated either around the time of immunization with collagen or post-immunization. For example, in some groups, EVs may be administered at the same time as immunization (day 1), or EVs may be administered upon first signs of disease, or upon the onset of severe symptoms. EVs are administered at varied doses and at defined intervals.


For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other groups of mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, or nasal route administration.


Some groups of mice may be treated with additional anti-inflammatory agent(s) or CIA therapeutic(s) (e.g. anti-CD154, blockade of members of the TNF family, Vitamin D, or other treatment), and/or an appropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics.


At various timepoints, serum samples are obtained to assess levels of anti-chick and anti-mouse CII IgG antibodies using a standard ELISA (Batsalova et al. Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains. Arthritis Res Ther. 2012. 14(6): R237). Also, some mice are sacrificed and sites of inflammation (e.g. synovium), lymph nodes, or other tissues may be removed for ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. The synovium and synovial fluid are analyzed for plasma cell infiltration and the presence of antibodies using techniques known in the art. In addition, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions to examine the profiles of the cellular infiltrates. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+synovium-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be rechallenged with a disease trigger (e.g. activated re-injection with CIA-inducing peptides). Mice are analyzed for susceptibility to disease and CIA severity following rechallenge.


Example 18: EVs in a Mouse Model of Colitis

Dextran sulfate sodium (DSS)-induced colitis is a well-studied animal model of colitis, as reviewed by Randhawa et al. (A review on chemical-induced inflammatory bowel disease models in rodents. Korean J Physiol Pharmacol. 2014. 18(4): 279-288; see also Chassaing et al. Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol. 2014 Feb. 4; 104: Unit 15.25).


EVs are tested for their efficacy in a mouse model of DSS-induced colitis, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory agents.


Groups of mice are treated with DSS to induce colitis as known in the art (Randhawa et al. 2014; Chassaing et al. 2014; see also Kim et al. Investigating intestinal inflammation in DSS-induced model of IBD. J Vis Exp. 2012. 60: 3678). For example, male 6-8 week old C57Bl/6 mice are obtained from Charles River Labs, Taconic, or other vendor. Colitis is induced by adding 3% DSS (MP Biomedicals, Cat. #0260110) to the drinking water. Some mice do not receive DSS in the drinking water and serve as naïve controls. Some mice receive water for five (5) days. Some mice may receive DSS for a shorter duration or longer than five (5) days. Mice are monitored and scored using a disability activity index known in the art based on weight loss (e.g. no weight loss (score 0); 1-5% weight loss (score 1); 5-10% weight loss (score 2)); stool consistency (e.g. normal (score 0); loose stool (score 2); diarrhea (score 4)); and bleeding (e.g. no blood (score 0), hemoccult positive (score 1); hemoccult positive and visual pellet bleeding (score 2); blood around anus, gross bleeding (score 4).


Treatment with EVs is initiated at some point, either on day 1 of DSS administration, or sometime thereafter. For example, EVs may be administered at the same time as DSS initiation (day 1), or they may be administered upon the first signs of disease (e.g. weight loss or diarrhea), or during the stages of severe colitis. Mice are observed daily for weight, morbidity, survival, presence of diarrhea and/or bloody stool.


EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route administration.


Some groups of mice may be treated with additional anti-inflammatory agent(s) (e.g. anti-CD154, blockade of members of the TNF family, or other treatment), and/or an appropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some mice receive DSS without receiving antibiotics beforehand.


At various timepoints, mice undergo video endoscopy using a small animal endoscope (Karl Storz Endoskipe, Germany) under isoflurane anesthesia. Still images and video are recorded to evaluate the extent of colitis and the response to treatment. Colitis is scored using criteria known in the art. Fecal material is collected for study.


At various timepoints, mice are sacrificed and the colon, small intestine, spleen, and lymph nodes (e.g. mesenteric lymph nodes) are collected. Additionally, blood is collected into serum separation tubes. Tissue damage is assessed through histological studies that evaluate, but are not limited to, crypt architecture, degree of inflammatory cell infiltration, and goblet cell depletion.


The gastrointestinal (GI) tract, lymph nodes, and/or other tissues may be removed for ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. For example, tissues are harvested and may be dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+ GI tract-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be rechallenged with a disease trigger. Mice are analyzed for susceptibility to colitis severity following rechallenge.


Example 19: EVs in a Mouse Model of Delayed-Type Hypersensitivity (DTH)

Delayed-type hypersensitivity (DTH) is an animal model of atopic dermatitis (or allergic contact dermatitis), as reviewed by Petersen et al. (In vivo pharmacological disease models for psoriasis and atopic dermatitis in drug discovery. Basic & Clinical Pharm & Toxicology. 2006. 99(2): 104-115; see also Irving C. Allen (ed.) Mouse Models of Innate Immunity: Methods and Protocols, Methods in Molecular Biology, 2013. vol. 1031, DOI 10.1007/978-1-62703-481-4_13). It can be induced in a variety of mouse and rat strains using various haptens or antigens, for example an antigen emulsified with an adjuvant. DTH is characterized by sensitization as well as an antigen-specific T cell-mediated reaction that results in erythema, edema, and cellular infiltration—especially infiltration of antigen presenting cells (APCs), eosinophils, activated CD4+ T cells, and cytokine-expressing Th2 cells.


Generally, mice are primed with an antigen administered in the context of an adjuvant (e.g. Complete Freund's Adjuvant) in order to induce a secondary (or memory) immune response measured by swelling and antigen-specific antibody titer.


EVs are tested for their efficacy in the mouse model of DTH, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, 6-8 week old C57Bl/6 mice are obtained from Taconic (Germantown, NY), or other vendor. Groups of mice are administered four subcutaneous (s.c.) injections at four sites on the back (upper and lower) of antigen (e.g. Ovalbumin (OVA)) in an effective dose (50 μl total volume per site). For a DTH response, animals are injected intradermally (i.d.) in the ears under ketamine/xylazine anesthesia (approximately 50 mg/kg and 5 mg/kg, respectively). Some mice serve as control animals. Some groups of mice are challenged with 10 μl per ear (vehicle control (0.01% DMSO in saline) in the left ear and antigen (21.2 ug (12 nmol) in the right ear) on day 8. To measure ear inflammation, the ear thickness of manually restrained animals is measured using a Mitutoyo micrometer. The ear thickness is measured before intradermal challenge as the baseline level for each individual animal. Subsequently, the ear thickness is measured two times after intradermal challenge, at approximately 24 hours and 48 hours (i.e. days 9 and 10).


Treatment with EVs is initiated at some point, either around the time of priming or around the time of DTH challenge. For example, EVs may be administered at the same time as the subcutaneous injections (day 0), or they may be administered prior to, or upon, intradermal injection. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, topical administration, intradermal (i.d.) injection, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 0), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×10+ and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, i.d. injection, topical administration, or nasal route administration.


Mice were injected with KLH and CFA i.d at 4 locations along the back (50 ug per mouse of KLH prepared in a 1:1 ratio with CFA in a total volume of 50 μl per site). Mice were dosed for 9 days as follows; 1) oral administration of anaerobic PBS (vehicle); 2) oral administration of 10 mg Prevotella histicola; 3) oral administration of 100 ug P. histicola-derived EVs; 4) i.p. administration of PBS; 5) i.p. administration of Dexamethasone (positive control); and 6) i.p. administration of 10 ug Prevotella histicola-derived EVs. For the EVs, total protein was measured using Bio-rad assays (Cat #5000205) performed per manufacturer's instructions. At 24 and 48 hours post-challenge with 10 ug of KLH (10 μl volume), groups receiving Prevotella histicola (live cells) or Prevotella histicola-derived EVs, in both the oral and i.p administration groups, exhibited less inflammation than the vehicle groups (FIGS. 3A and 3B). A dose dependent DTH response following i.p. injection of Prevotella histicola-derived EVs at 10 μg, 3 μg, 1 μg, and 0.1 μg was observed in reducing antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a KLH-based delayed type hypersensitivity mouse model (FIG. 3C).


Mice were injected with KLH and CFA i.d at 4 locations along the back (50 ug per mouse of KLH prepared in a 1:1 ratio with CFA in a total volume of 50 μl per site). Mice were dosed for 9 days as follows; 1) oral administration of anaerobic PBS (vehicle); 2) oral administration of 10 mg Prevotella histicola; 3) oral administration of 1×109 CFU Prevotella histicola biomass; 4) oral administration of 2.09×108 CFU Prevotella melanogenica biomass; 5) oral administration of 100 ug P. histicola-derived EVs; 6) oral administration of 100 ug P. melanogenica-derived EVs; and 7) i.p. administration of Dexamethasone (positive control). For the EVs, total protein was measured using Bio-rad assays (Cat #5000205) performed per manufacturer's instructions. At 24 and 48 hours post-challenge with 10 ug of KLH (10 μl volume), groups receiving Prevotella histicola (live cells) or Prevotella histicola-derived EVs exhibited less inflammation than the vehicle groups (FIGS. 8A and 8B). At 24 and 48 hours post-challenge with 10 ug of KLH (10 μl volume), the group receiving Prevotella melanogenica-derived EVs exhibited less inflammation than the vehicle groups and the group receiving Prevotella melanogenica (live cells) (FIGS. 8A and 8B).


The test formulations were prepared for KLH-based delayed type hypersensitivity model. The DTH model provides an in vivo mechanism to study the cell-mediated immune response, and resulting inflammation, following exposure to a specific antigen to which the mice have been sensitized. Several variations of the DTH model have been used and are well known in the art (Irving C. Allen (ed.). Mouse Models of Innate Immunity: Methods and Protocols, Methods in Molecular Biology. Vol. 1031, DOI 10.1007/978-1-62703-481-4_13, Springer Science+Business Media, LLC 2013). For example, the emulsion of Keyhole Limpet Hemocyanin (KLH) and Complete Freund's Adjuvant (CFA) are prepared freshly on the day of immunization (day 0). To this end, 8 mg of KLH powder is weighed and is thoroughly re-suspended in 16 mL saline. An emulsion is prepared by mixing the KLH/saline with an equal volume of CFA solution (e.g. 10 mL KLH/saline+10 mL CFA solution) using syringes and a luer lock connector. KLH and CFA is mixed vigorously for several minutes to form a white-colored emulsion to obtain maximum stability. A drop test is performed to check if a homogenous emulsion is obtained, mixing is continued until an intact drop remains visible in the water.


On day 0, C57Bl/6J female mice, approximately 7 weeks old, were primed with KLH antigen in CFA by subcutaneous immunization (4 sites, 50 μL per site).


Dexamethasone, a corticosteroid, is a known anti-inflammatory that ameliorates DTH reactions in mice, and serves as a positive control for suppressing inflammation in this model (Taube and Carlsten, Action of dexamethasone in the suppression of delayed-type hypersensitivity in reconstituted SCID mice. Inflamm Res. 2000. 49(10): 548-52). For the positive control group, a stock solution of 17 mg/mL of Dexamethasone was prepared on Day 0 by diluting 6.8 mg Dexamethasone in 400 μL 96% ethanol. For each day of dosing, a working solution is prepared by diluting the stock solution 100× in sterile PBS to obtain a final concentration of 0.17 mg/mL in a septum vial for intraperitoneal dosing. Dexamethasone-treated mice received 100 μL Dexamethasone i.p. (5 mL/kg of a 0.17 mg/mL solution). Frozen sucrose served as the negative control (vehicle). Veillonella Strains were dosed at 1×1010 CFU p.o. daily. Dexamethasone (positive control), vehicle (negative control), and Bifidobacterium animalis lactis (10 mg powder) were dosed daily.


On day 8, mice were challenged intradermally (i.d.) with 10 μg KLH in saline (in a volume of 10 μL) in the left ear. Inflammatory responses were measured using methods known in the art. Ear pinna thickness was measured at 24 hours following antigen challenge (FIG. 11). As determined by ear thickness, Veillonella Strains were efficacious at suppressing inflammation compared to mice that received vehicle alone (comparable to Dexamethasone treatment).


The efficacy of Veillonella strains may be studied further using varied timing and varied doses. For instance, treatment with a Veillonella bacterial composition may be initiated at some point, either around the time of priming or around the time of DTH challenge. For example, Veillonella (1×109 CFU per mouse per day) may be administered at the same time as the subcutaneous injections (day 0), or administered prior to, or upon, intradermal injection. Veillonella strains may be administered at varied doses and at defined intervals, and in various combinations. For example, some mice are intravenously injected with Veillonella at a range of between 1×104 and 5×109 bacterial cells per mouse. Some mice receive a mixture of Strains. While some mice will receive a Veillonella through i.v. injection, other mice may receive a Veillonella through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, topical administration, intradermal (i.d.) injection, or other means of administration. Some mice may receive a Veillonella every day (e.g. starting on day 0), while others may receive a Veillonella at alternative intervals (e.g. every other day, or once every three days). The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


Some groups of mice may be treated with anti-inflammatory agent(s) (e.g. anti-CD154, blockade of members of the TNF family, or other treatment), and/or an appropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics.


At various timepoints, serum samples are taken. Other groups of mice are sacrificed and lymph nodes, spleen, mesenteric lymph nodes (MLN), the small intestine, colon, and other tissues may be removed for histology studies, ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. Some mice are exsanguinated from the orbital plexus under O2/CO2 anesthesia and ELISA assays performed.


Tissues may be dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


Mice were primed and challenged with KLH as described above and, following measurement of the ear swelling at 48 hours, mice were sacrificed.


Ears were removed from the sacrificed animals and placed in cold EDTA-free protease inhibitor cocktail (Roche). Ears were homogenized using bead disruption and supernatants analyzed for IL-1ß by Luminex kit (EMD Millipore) as per manufacturer's instructions. Mice that were treated with 10 μg P. histicola EVs (i.p.) showed levels of IL-1B comparable to that seen in the Dexamethasone group (positive control). (FIG. 3D). P. histicola-derived EVs are capable of suppressing pro-inflammatory cytokines.


In addition, cervical lymph nodes were dissociated through a cell strainer, washed, and stained for FoxP3 (PE-FJK-16s) and CD25 (FITC-PC61.5) using methods known in the art (FIG. 3E). Mice that were treated with 10 μg P. histicola EVs (i.p.) showed an increase in Tregs in the cervical lymph nodes relative to naïve mice (negative control), and comparable to the Dexamethasone group (positive control). P. histicola-derived EVs are capable of inducing Tregs in draining lymph nodes of challenged mice.


In order to examine the impact and longevity of DTH protection, rather than being sacrificed, some mice may be rechallenged with the challenging antigen (e.g. OVA). Mice are analyzed for susceptibility to DTH and severity of response.


Example 20: EVs in a Mouse Model of Type 1 Diabetes (TID)

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system targets the islets of Langerhans of the pancreas, thereby destroying the body's ability to produce insulin.


There are various models of animal models of TID, as reviewed by Belle et al. (Mouse models for type 1 diabetes. Drug Discov Today Dis Models. 2009; 6(2): 41-45; see also Aileen J F King. The use of animal models in diabetes research. Br J Pharmacol. 2012 June; 166(3): 877-894. There are models for chemically-induced TID, pathogen-induced TID, as well as models in which the mice spontaneously develop TID.


EVs are tested for their efficacy in a mouse model of TID, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments.


Depending on the method of TID induction and/or whether TID development is spontaneous, treatment with EVs is initiated at some point, either around the time of induction or following induction, or prior to the onset (or upon the onset) of spontaneously-occurring TID. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day, while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×10+ and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route administration.


Some groups of mice may be treated with additional treatments and/or an appropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics.


Blood glucose is monitored biweekly prior to the start of the experiment. At various timepoints thereafter, nonfasting blood glucose is measured. At various timepoints, mice are sacrificed and site the pancreas, lymph nodes, or other tissues may be removed for ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. For example, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified tissue-infiltrating immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression. Antibody production may also be assessed by ELISA.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be rechallenged with a disease trigger, or assessed for susceptibility to relapse. Mice are analyzed for susceptibility to diabetes onset and severity following rechallenge (or spontaneously-occurring relapse).


Example 21: EVs in a Mouse Model of Primary Sclerosing Cholangitis (PSC)

Primary Sclerosing Cholangitis (PSC) is a chronic liver disease that slowly damages the bile ducts and leads to end-stage cirrhosis. It is associated with inflammatory bowel disease (IBD).


There are various animal models for PSC, as reviewed by Fickert et al. (Characterization of animal models for primary sclerosing cholangitis (PSC). J Hepatol. 2014 June 60(6): 1290-1303; see also Pollheimer and Fickert. Animal models in primary biliary cirrhosis and primary sclerosing cholangitis. Clin Rev Allergy Immunol. 2015 June 48(2-3): 207-17). Induction of disease in PSC models includes chemical induction (e.g. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholangitis), pathogen-induced (e.g. Cryptosporidium parvum), experimental biliary obstruction (e.g. common bile duct ligation (CBDL)), and transgenic mouse model of antigen-driven biliary injury (e.g. Ova-Bil transgenic mice). For example, bile duct ligation is performed as described by Georgiev et al. (Characterization of time-related changes after experimental bile duct ligation. Br J Surg. 2008. 95(5): 646-56), or disease is induced by DCC exposure as described by Fickert et al. (A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. Am J Path. Vol 171(2): 525-536.


EVs are tested for their efficacy in a mouse model of PSC, either alone or in combination with whole bacterial cells, with or without the addition of some other therapeutic agent.


DCC-Induced Cholangitis


For example, 6-8 week old C57bl/6 mice are obtained from Taconic or other vendor. Mice are fed a 0.1% DCC-supplemented diet for various durations. Some groups receive DCC-supplement food for 1 week, others for 4 weeks, others for 8 weeks. Some groups of mice may receive a DCC-supplemented diet for a length of time and then be allowed to recover, thereafter receiving a normal diet. These mice may be studied for their ability to recover from disease and/or their susceptibility to relapse upon subsequent exposure to DCC. Treatment with EVs is initiated at some point, either around the time of DCC-feeding or subsequent to initial exposure to DCC. For example, EVs may be administered on day 1, or they may be administered sometime thereafter. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, Or 15 ug/mouse. Other mice may receive 25, 50, 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through i.p. injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen), and administered, or they may be irradiated or heat-killed prior to administration. For example, some groups of mice may receive between 1×10+ and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route administration. Some groups of mice may be treated with additional agents and/or an appropriate control (e.g. vehicle or antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics. At various timepoints, serum samples are analyzed for ALT, AP, bilirubin, and serum bile acid (BA) levels.


At various timepoints, mice are sacrificed, body and liver weight are recorded, and sites of inflammation (e.g. liver, small and large intestine, spleen), lymph nodes, or other tissues may be removed for ex vivo histolomorphological characterization, cytokine and/or flow cytometric analysis using methods known in the art (see Fickert et al. Characterization of animal models for primary sclerosing cholangitis (PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts are stained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues are stained for histological examination, while others are dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well as adhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+bile duct-infiltrated immune cells obtained ex vivo.


Liver tissue is prepared for histological analysis, for example, using Sirius-red staining followed by quantification of the fibrotic area. At the end of the treatment, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, and to determine Bilirubin levels. The hepatic content of Hydroxyproline can be measured using established protocols. Hepatic gene expression analysis of inflammation and fibrosis markers may be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, MCP-1, alpha-SMA, Collla1, and TIMP-. Metabolite measurements may be performed in plasma, tissue and fecal samples using established metabolomics methods. Finally, immunohistochemistry is carried out on liver sections to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be rechallenged with DCC at a later time. Mice are analyzed for susceptibility to cholangitis and cholangitis severity following rechallenge.


BDL-Induced Cholangitis


Alternatively, EVs are tested for their efficacy in BDL-induced cholangitis. For example, 6-8 week old C57Bl/6J mice are obtained from Taconic or other vendor. After an acclimation period the mice are subjected to a surgical procedure to perform a bile duct ligation (BDL). Some control animals receive a sham surgery. The BDL procedure leads to liver injury, inflammation and fibrosis within 7-21 days.


Treatment with EVs is initiated at some point, either around the time of surgery or some time following the surgery. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through i.p. injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. They bacterial cells may be harvested fresh (or frozen), and administered, or they may be irradiated or heat-killed prior to administration. For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route administration. Some groups of mice may be treated with additional agents and/or an appropriate control (e.g. vehicle or antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics. At various timepoints, serum samples are analyzed for ALT, AP, bilirubin, and serum bile acid (BA) levels.


At various timepoints, mice are sacrificed, body and liver weight are recorded, and sites of inflammation (e.g. liver, small and large intestine, spleen), lymph nodes, or other tissues may be removed for ex vivo histolomorphological characterization, cytokine and/or flow cytometric analysis using methods known in the art (see Fickert et al. Characterization of animal models for primary sclerosing cholangitis (PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts are stained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues are stained for histological examination, while others are dissociated using dissociation enzymes according to the manufacturer's instructions. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSFIR, PD-L1, Gr-1, F4/80), as well as adhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+bile duct-infiltrated immune cells obtained ex vivo.


Liver tissue is prepared for histological analysis, for example, using Sirius-red staining followed by quantification of the fibrotic area. At the end of the treatment, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, and to determine Bilirubin levels. The hepatic content of Hydroxyproline can be measured using established protocols. Hepatic gene expression analysis of inflammation and fibrosis markers may be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, MCP-1, alpha-SMA, Collla1, and TIMP-. Metabolite measurements may be performed in plasma, tissue and fecal samples using established metabolomics methods. Finally, immunohistochemistry is carried out on liver sections to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be analyzed for recovery.


Example 22: EVs in a Mouse Model of Nonalcoholic Steatohepatitis (NASH)

Nonalcoholic Steatohepatitis (NASH) is a severe form of Nonalcoholic Fatty Liver Disease (NAFLD), where buildup of hepatic fat (steatosis) and inflammation lead to liver injury and hepatocyte cell death (ballooning).


There are various animal models of NASH, as reviewed by Ibrahim et al. (Animal models of nonalcoholic steatohepatitis: Eat, Delete, and Inflame. Dig Dis Sci. 2016 May. 61(5): 1325-1336; see also Lau et al. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances 2017 January 241(1): 36-44).


EVs are tested for their efficacy in a mouse model of NASH, either alone or in combination with whole bacterial cells, with or without the addition of another therapeutic agent. For example, 8-10 week old C57Bl/6J mice, obtained from Taconic (Germantown, NY), or other vendor, are placed on a methionine choline deficient (MCD) diet for a period of 4-8 weeks during which NASH features develop, including steatosis, inflammation, ballooning and fibrosis.



Prevotella histicola bacterial cells and P. histicola-derived EVs are tested for their efficacy in a mouse model of NASH, either alone or in combination with each other, in varying proportions, with or without the addition of another therapeutic agent. For example, 8 week old C57Bl/6J mice, obtained from Charles River (France), or other vendor, were acclimated for a period of 5 days, randomized intro groups of 10 mice based on body weight, and placed on a methionine choline deficient (MCD) diet for example A02082002B from Research Diets (USA), for a period of 4 weeks during which NASH features developed, including steatosis, inflammation, ballooning and fibrosis. Control chow mice were fed a normal chow diet, for example RMI (E) 801492 from SDS Diets (UK). Control chow, MCD diet, and water were provided ad libitum.


Treatment with frozen, live P. histicola was initiated in day 1 of MCD diet for some mice and continued for 28 consecutive days. Some MCD diet mice were administered bacterial cells through daily oral gavage of 100 μl of a suspension containing 1.47×109 bacterial cells. Control chow and some MCD diet mice remained untreated, while some MCD diet mice were administered daily with a vehicle solution, through daily oral gavage, for 28 days. Some MCD diet mice were administered the reference compound and FXR agonist, obeticholic acid (OCA; positive control), at a dose of 30 mg/kg, through daily oral gavage, for 28 days. At the end of the treatment (day 28), mice are sacrificed and liver, small intestine, lumenal contents, blood, and feces, were removed for ex vivo histological, biochemical, molecular or cytokine and/or flow cytometry analysis using methods known in the art. For example, 0.5 cm3 liver samples were stored in formalin for 24 hours and then in ethanol at 4° C., prior to hematoxylin/eosin (H&E) and Sirius Red staining, and determination of NASH activity score (NAS). Histological analysis and scoring was conducted at Histalim (Montpelier, France) in a blinded manner. Slides containing one hepatic lobe section stained with either H&E or Sirius red were digitized using a NanoZoomer and visualized using NDP viewer, both from Hamamatsu (Japan). Each section was evaluated and scored individually. A NAS scoring system adapted from Kleiner et al. (Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005 June 41(6): 1313-1321) was used to determine the degree of steatosis (scored 0-3), lobular inflammation (scored 0-3), hepatocyte ballooning (scored 0-3), and fibrosis (scored 0-4). An individual mouse NAS score was calculated by summing the score for steatosis, inflammation, ballooning, and fibrosis (scored 0-13). In addition, the levels of plasma AST and ALT were determined using a Pentra 400 instrument from Horiba (USA), according to manufacturer's instructions. The levels of hepatic total cholesterol, triglycerides, fatty acids, alanine aminotransferase, and aspartate aminotransferase were also determined using methods known in the art.


In mice receiving the MCD (NASH-inducing) diet, orally administered P. histicola was efficacious in reducing the NAS score compared to vehicle and no treatment groups (negative controls) (FIG. 4). P. histicola reduced steatosis (FIG. 5A), inflammation (FIG. 5B and FIG. 5C), and ballooning (FIG. 5D), as well as hepatic total cholesterol (FIG. 6). P. histicola also reduced the fibrosis score in treated mice (FIG. 7A and FIG. 7B).



FIG. 9A shows P. histicola effect on hepatic free fatty acids in mice that were fed an MCD diet, FIG. 9B shows P. histicola effect on hepatic total cholesterol in mice that were fed an MCD diet, FIG. 9C shows P. histicola effect on hepatic triglycerides in mice that were fed an MCD diet, FIG. 9D shows P. histicola and P. melanogenica effect on alanine aminotransferase in mice that were fed an MCD diet, FIG. 9E shows P. histicola and P. melanogenica effect on aspartate aminotransferase in mice that were fed an MCD diet.


In mice receiving the MCD (NASH-inducing) diet, orally administered P. histicola and P. melanogenica was efficacious in reducing the NAS score compared to vehicle and no treatment groups (negative controls) (FIGS. 10A and 10B).


In other studies, hepatic gene expression analysis of inflammation, fibrosis, steatosis, ER stress, or oxidative stress markers may be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, IL-1β, TNF-α, MCP-1, α-SMA, Coll1a1, CHOP, and NRF2.


Treatment with EVs is initiated at some point, either at the beginning of the diet, or at some point following diet initiation (for example, one week after). For example, EVs may be administered starting in the same day as the initiation of the MCD diet. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route administration. Some groups of mice may be treated with additional NASH therapeutic(s) (e.g., FXR agonists, PPAR agonists, CCR2/5 antagonists or other treatment) and/or appropriate control at various timepoints and effective doses.


At various timepoints and/or at the end of the treatment, mice are sacrificed and liver, intestine, blood, feces, or other tissues may be removed for ex vivo histological, biochemical, molecular or cytokine and/or flow cytometry analysis using methods known in the art. For example, liver tissues are weighed and prepared for histological analysis, which may comprise staining with H&E, Sirius Red, and determination of NASH activity score (NAS). At various timepoints, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, using standards assays. In addition, the hepatic content of cholesterol, triglycerides, or fatty acid acids can be measured using established protocols. Hepatic gene expression analysis of inflammation, fibrosis, steatosis, ER stress, or oxidative stress markers may be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, IL-6, MCP-1, alpha-SMA, Coll1a1, CHOP, and NRF2. Metabolite measurements may be performed in plasma, tissue and fecal samples using established biochemical and mass-spectrometry-based metabolomics methods. Serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+bile duct-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on liver or intestine sections to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.


In order to examine the impact and longevity of disease protection, rather than being sacrificed, some mice may be analyzed for recovery.


Example 23: EVs in a Mouse Model of Psoriasis

Psoriasis is a T-cell-mediated chronic inflammatory skin disease. So-called “plaque-type” psoriasis is the most common form of psoriasis and is typified by dry scales, red plaques, and thickening of the skin due to infiltration of immune cells into the dermis and epidermis. Several animal models have contributed to the understanding of this disease, as reviewed by Gudjonsson et al. (Mouse models of psoriasis. J Invest Derm. 2007. 127: 1292-1308; see also van der Fits et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J. Immunol. 2009 May 1. 182(9): 5836-45).


Psoriasis can be induced in a variety of mouse models, including those that use transgenic, knockout, or xenograft models, as well as topical application of imiquimod (IMQ), a TLR7/8 ligand.


EVs are tested for their efficacy in the mouse model of psoriasis, either alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, 6-8 week old C57Bl/6 or Balb/c mice are obtained from Taconic (Germantown, NY), or other vendor. Mice are shaved on the back and the right ear. Groups of mice receive a daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals). The dose is applied to the shaved areas for 5 or 6 consecutive days. At regular intervals, mice are scored for erythema, scaling, and thickening on a scale from 0 to 4, as described by van der Fits et al. (2009). Mice are monitored for ear thickness using a Mitutoyo micrometer.


Treatment with EVs is initiated at some point, either around the time of the first application of IMQ, or something thereafter. For example, EVs may be administered at the same time as the subcutaneous injections (day 0), or they may be administered prior to, or upon, application. EVs are administered at varied doses and at defined intervals. For example, some mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mg of EVs per mouse. While some mice receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, nasal route administration, oral gavage, topical administration, intradermal (i.d.) injection, subcutaneous (s.c.) injection, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 0), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened. The bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.


For example, some groups of mice may receive between 1×104 and 5×109 bacterial cells in an administration separate from, or comingled with, the EV administration. As with the EVs, bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, i.d. injection, s.c. injection, topical administration, or nasal route administration.


Some groups of mice may be treated with anti-inflammatory agent(s) (e.g. anti-CD154, blockade of members of the TNF family, or other treatment), and/or an appropriate control (e.g. vehicle or control antibody) at various timepoints and at effective doses.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some immunized mice are treated without receiving antibiotics.


At various timepoints, samples from back and ear skin are taken for cryosection staining analysis using methods known in the art. Other groups of mice are sacrificed and lymph nodes, spleen, mesenteric lymph nodes (MLN), the small intestine, colon, and other tissues may be removed for histology studies, ex vivo histological, cytokine and/or flow cytometric analysis using methods known in the art. Some tissues may be dissociated using dissociation enzymes according to the manufacturer's instructions. Cryosection samples, tissue samples, or cells obtained ex vivo are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSFIR, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out on immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+ skin-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on various tissue sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


In order to examine the impact and longevity of psoriasis protection, rather than being sacrificed, some mice may be studied to assess recovery, or they may be rechallenged with IMQ. The groups of rechallenged mice are analyzed for susceptibility to psoriasis and severity of response.


Example 24: Manufacturing Conditions

Enriched media is used to grow and prepare the bacterium for in vitro and in vivo use. For example, media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins. Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.). Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested. Alternatively, media may be prepared and the selected bacterium grown as shown by Saarela et al., J. Applied Microbiology. 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of the selected bacterium produced without milk-based ingredients.


At large scale, the media is sterilized. Sterilization may be by Ultra High Temperature (UHT) processing. The UHT processing is performed at very high temperature for short periods of time. The UHT range may be from 135-180° C. For example, the medium may be sterilized from between 10 to 30 seconds at 135° C.


Inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored. For example, the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume. Depending on the application and need for material, bioreactor volume can be at least 2L, 10L, 80L, 100L, 250L, 1000L, 2500L, 5000L, 10,000L.


Before the inoculation, the bioreactor is prepared with medium at desired pH, temperature, and oxygen concentration. The initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0. During the fermentation, the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide. The temperature may be controlled from 25° C. to 45° C., for example at 37° C. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8 mg/L to Omg/L. For example, nitrogen or gas mixtures (N2, CO2, and H2) may be used in order to establish anaerobic conditions. Alternatively, no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium. Depending on strain and inoculum size, the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.


Reviving microbes from a frozen state may require special considerations. Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material. The kinetics of transfer or passage of seed material to fresh medium, for the purposes of increasing the seed volume or maintaining the microbial growth state, may be influenced by the current state of the microbes (ex. exponential growth, stationary growth, unstressed, stressed).


Inoculation of the production fermenter(s) can impact growth kinetics and cellular activity. The initial state of the bioreactor system must be optimized to facilitate successful and consistent production. The fraction of seed culture to total medium (e.g. a percentage) has a dramatic impact on growth kinetics. The range may be 1-5% of the fermenter's working volume. The initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration.


Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc. Optimal temperature range for growth may vary with strain. The range may be 20-40° C. Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Microbes may have alternate kinetics when excess nutrients are available.


The state of microbes at the end of a fermentation and during harvesting may impact cell survival and activity. Microbes may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and downstream processing. A change in temperature (often reducing to 20-5° C.) may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter. Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1-2 points can improve effectiveness of concentration but can also be detrimental to cells. Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream.


Separation methods and technology may impact how efficiently microbes are separated from the culture medium. Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1-2 points may improve effectiveness of concentration but can also be detrimental to cells. Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Microbes may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g-minutes to successfully centrifuge. Excipients can be added before after separation. Excipients can be added for cryo protection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants. Prior to lyophilization, droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.


Harvesting can be performed by continuous centrifugation. Product may be resuspended with various excipients to a desired final concentration. Excipients can be added for cryo protection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants. Prior to lyophilization, droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.


Lyophilization of material, including live bacteria, begins with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime. During the secondary drying phase, product bound water molecules are removed. Here, the temperature is raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material. The pressure may also be lowered further to enhance desorption during this stage. After the freeze-drying process is complete, the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants.


Example 25: A Mouse Melanoma Model

Female 6-8 week old C57Bl/6 mice are obtained from Taconic (Germantown, NY). 100,000 B16-F10 (ATCC CRL-6475) tumor cells are resuspended in sterile PBS containing 50% Matrigel and inoculated in a 100 μl final volume into one hind flank (the first flank) of each mouse. Treatment with Veillonella Strains is initiated at some point following tumor cell inoculation at varied doses and at defined intervals. For example, some mice receive between 1-5×10{circumflex over ( )}9 CFU (100 μl final volume) per dose. Possible routes of administration include oral gavage (p.o.), intravenous injection, intratumoral injection (IT) or peritumoral or subtumoral or subcutaneous injection. In order to assess the systemic anti-tumoral effects of Veillonella treatment, additional mice may be inoculated with tumor cells in the contralateral (untreated, second) flank prior to IT, peritumoral, or subtumoral treatment with Veillonella in the first flank.


Some mice may receive Veillonella (p.o.) on day 1 (the day following tumor cell injection). Other mice may receive seven (7) consecutive doses of a bacterial strain (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm3) and treatment is then initiated accordingly. For example, when tumor volumes reach an average of 100 mm3 (approximately 10-12 days following tumor cell inoculation), animals are distributed into groups and treated with either vehicle or a bacterial strain (p.o. or IT). Some additional groups of mice may be treated with an additional cancer therapeutic or appropriate control antibody. One example of a cancer therapeutic that may be administered is an inhibitor of an immune checkpoint, for example anti-PD-1, anti-PD-L1, or other treatment that blocks the binding of an immune checkpoint to its ligand(s). Checkpoint inhibitors anti-PD-1 and anti-PD-L1 may be formulated in PBS and administered intraperitoneally (i.p.) in effective doses. For example, mice are given 100 ug of anti-PD-1 (i.p.) every four days starting on day 1, and continuing for the duration of the study.


In addition, some mice are treated with antibiotics prior to treatment. For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0 g/L) and amphotericin B (0.2 g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment. Some mice are inoculated with tumor cells without receiving prior treatment with antibiotics.


At various timepoints, mice are sacrificed and tumors, lymph nodes, or other tissues may be removed for ex vivo flow cytometric analysis using methods known in the art. For example, tumors are dissociated using a Miltenyi tumor dissociation enzyme cocktail according to the manufacturer's instructions. Tumor weights are recorded and tumors are chopped then placed in 15 ml tubes containing the enzyme cocktail and placed on ice. Samples are then placed on a gentle shaker at 37° C. for 45 minutes and quenched with up to 15 ml complete RPMI. Each cell suspension is strained through a 70 μm filter into a 50 ml falcon tube and centrifuged at 1000 rpm for 10 minutes. Cells are resuspended in FACS buffer and washed to remove remaining debris. If necessary, samples are strained again through a second 70 μm filter into a new tube. Cells are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzed include pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSFIR, PD-L1, Gr-1). In addition to immunophenotyping, serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may be carried out immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+ tumor-infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is carried out on tumor sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.


Rather than being sacrificed, some mice may be rechallenged with tumor cell injection into the contralateral flank (or other area) to determine the impact of the immune system's memory response on tumor growth.


In mice receiving the MCD (NASH-inducing) diet, orally administered Veillonella was efficacious in reducing the NAS score compared to vehicle and no treatment groups (negative controls) (FIG. 16). Veillonella reduced the fibrosis score in treated mice (FIG. 17). Veillonella reduced hepatic total cholesterol (FIG. 18) and hepatic Triglycerides (FIG. 19).


Example 26: A Colorectal Carcinoma Model

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown, NY). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% Matrigel. CT-26 tumor cells were subcutaneously injected into one hind flank of each mouse. When tumor volumes reached an average of 100 mm3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups: 1) Vehicle; 2) Veillonella Strains 3) anti-PD-1 antibody. Antibodies were administered intraperitoneally (i.p.) at 200 μg/mouse (100 μl final volume) every four days, starting on day 1, for a total of 3 times (Q4D×3) and Veillonella EVs (5 μg) were intravenously (i.v.) injected every third day, starting on day 1 for a total of 4 times (Q3D×4). Both Veillonella groups showed tumor growth inhibition greater than that seen in the anti-PD-1 group (FIGS. 12 and 13).


Another example, when tumor volumes reached an average of 100 mm3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups: 1) Vehicle; 2) Burkholderia pseudomallei; and 3) anti-PD-1 antibody. Antibodies were administered intraperitoneally (i.p.) at 200 μg/mouse (100 μl final volume) every four days, starting on day 1, and Burkholderia pseudomallei EVs (5 μg) were intravenously (i.v.) injected daily, starting on day 1 until the conclusion of the study. The Burkholderia pseudomallei group showed tumor growth inhibition greater than that seen in the anti-PD-1 group (FIGS. 20, and 21).


Another example, when tumor volumes reached an average of 100 mm3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups: 1) Vehicle; 2) Neisseria Meningitidis EVs isolated from the Bexsero® vaccine; and 3) anti-PD-1 antibody. Antibodies were administered intraperitoneally (i.p.) at 200 ug/mouse (100 μl final volume) every four days, starting on day 1, and Neisseria Meningitidis bacteria (about 1.1×102) were administered intraperitoneally (i.p.) daily, starting on day 1 until the conclusion of the study. The Neisseria Meningitidis group showed tumor growth inhibition greater than that seen in the anti-PD-1 group (FIGS. 22, and 23).









TABLE 4







Significance test comparing tumor volume in treatment


groups vs. control groups on day 11. T-test (two-tailed,


unpaired, welch-corrected) calculated in GraphPad.











Comparison
P value
Summary






IP Vehicle vs. Neisseria
0.0004
***




Meningitidis EVs.









Example 27: Efficacy of EVs Varies Based on Source Microbe, Dose, and Route of Administration

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown, NY). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% MATRIGEL®. CT-26 tumor cells were subcutaneously injected into one hind flank of each mouse. When tumor volumes reached an average of 100 mm3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups as highlighted in Table 5.









TABLE 5







Treatment Groups









Group
Treatment
Dose/Route/Schedule





1
IV Vehicle (PBS)
N/A/IV/Q3Dx4


2
PO Vehicle (sucrose)
N/A/PO/QD


3
Anti-PD-1
200 μg/IP/Q4Dx3


4

Veillonella parvula EV
10 μg/IV/Q3Dx4


5

Veillonella parvula EV
5 μg/IV/Q3Dx4


6

Veillonella parvula EV
2 μg/IV/Q3Dx4


7

Veillonella tobetsuensis EV
75 μg/PO. QD


8

Veillonella tobetsuensis EV
5 μg/IV/Q3Dx4









As noted in the table, antibodies were administered intraperitoneally (i.p.) at 200 μg/mouse (100 μl final volume) every four days, starting on day 1, for a total of 3 times (Q4D×3) and EVs when administered intravenously (i.v.) were injected every third day, starting on day 1 for a total of 4 times (Q3D×4). The treatment groups administered by mouth (p.o.) were administered daily (QD). Efficacy of Veillonella EVs varies based on source microbe, dose, and route of administration (FIGS. 14 and 15).


INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.


EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims
  • 1. A method of treating an immune disorder in a subject comprising administering to the subject a pharmaceutical composition comprising isolated bacterial extracellular vesicles (EVs), wherein at least 90% of the total EV and bacteria particles in the pharmaceutical composition are EVs from Prevotella or Veillonella bacteria.
  • 2. The method of claim 1, wherein the pharmaceutical composition is administered orally.
  • 3. The method of claim 1, wherein the subject is a human.
  • 4. The method of claim 1, wherein the pharmaceutical composition is formulated as a capsule or tablet.
  • 5. The method of claim 1, wherein the immune disorder is psoriasis or atopic dermatitis.
  • 6. The method of claim 1, wherein the pharmaceutical composition comprises at least 1×106 isolated bacterial extracellular vesicles (EVs).
  • 7. The method of claim 1, wherein at least 95% of the total EV and bacteria particles in the pharmaceutical composition are EVs.
  • 8. The method of claim 1, wherein no more than 1% of the total EV and bacteria particles in the pharmaceutical composition are bacteria.
  • 9. The method of claim 1, wherein at least 95% of the total protein in the pharmaceutical composition is present in EVs.
  • 10. The method of claim 1, wherein no more than 1% of the total protein in the pharmaceutical composition is present in bacteria.
  • 11. The method of claim 1, wherein at least 95% of the total lipids in the pharmaceutical composition is present in EVs.
  • 12. The method of claim 1, wherein no more than 1% of the total lipids in the pharmaceutical composition is present in bacteria.
  • 13. The method of claim 1, wherein the pharmaceutical composition is free of bacteria.
  • 14. The method of claim 1, wherein the pharmaceutical composition comprises at least 5×106 isolated EVs.
  • 15. The method of claim 1, wherein the EVs are from a single strain of bacteria.
  • 16. The method of claim 1, wherein the EVs are from Prevotella histicola bacteria.
  • 17. The method of claim 1, wherein the EVs are from Veillonella tobetsuensis bacteria or Veillonella parvula bacteria.
  • 18. A method of treating psoriasis or atopic dermatitis in a subject comprising orally administering to the subject a pharmaceutical composition comprising isolated bacterial extracellular vesicles (EVs), wherein at least 90% of the total EV and bacteria particles in the pharmaceutical compositions are EVs from Prevotella histicola bacteria.
  • 19. A method of treating psoriasis or atopic dermatitis in a subject comprising orally administering to the subject a pharmaceutical composition comprising isolated bacterial extracellular vesicles (EVs), wherein at least 90% of the total EV and bacteria particles in the pharmaceutical compositions are EVs from Veillonella tobetsuensis bacteria or Veillonella parvula bacteria.
RELATED APPLICATIONS

This Application is a divisional of U.S. application Ser. No. 16/645,019, filed Mar. 6, 2020, which claims priority to national-stage application of International Application No. PCT/US2018/50211, filed Sep. 10, 2018, which claims the benefit of priority to U.S. Provisional Patent Applications having Ser. Nos. 62/556,015, filed Sep. 8, 2017, and 62/669,151, filed May 9, 2018, the contents of each of which are hereby incorporated herein by reference in their entirety.

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Related Publications (1)
Number Date Country
20230210913 A1 Jul 2023 US
Provisional Applications (2)
Number Date Country
62669151 May 2018 US
62556015 Sep 2017 US
Divisions (1)
Number Date Country
Parent 16645019 US
Child 18074122 US