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Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis
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  • Dharanesh Gangaiah,
  • Valerie Ryan,
  • Daphne van Hoesel,
  • Shrinivas Mane,
  • Enid McKinley,
  • Nallakannu Lakshmanan,
  • Nandakumar Reddy,
  • Edward Dolk,
  • Arvind Kumar
Dharanesh Gangaiah
Elanco Animal Health Inc

Corresponding Author:[email protected]

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Valerie Ryan
Elanco Animal Health Inc
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Daphne van Hoesel
QVQ Holding BV
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Shrinivas Mane
Elanco Animal Health Inc
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Enid McKinley
Elanco Animal Health Inc
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Nallakannu Lakshmanan
Elanco Animal Health Inc
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Nandakumar Reddy
Elanco Animal Health Inc
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Edward Dolk
QVQ Holding BV
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Arvind Kumar
Elanco Animal Health Inc
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Abstract

Necrotic enteritis (NE), caused by Clostridium perfringens, is an intestinal disease with devastating economic losses to the poultry industry. NE is a complex disease and predisposing factors that compromise gut integrity are required to facilitate C. perfringens proliferation and toxin production. NE is also characterized by drastic shifts in gut microbiota; C. perfringens is negatively correlated with Lactobacilli. Vaccines are only partially effective against NE and antibiotics suffer from the concern of resistance development. These strategies address only some aspects of NE pathogenesis. Thus, there is an urgent need for alternative strategies that address multiple aspects of NE biology. Here, we developed Limosilactobacillus (Lactobacillus) reuteri vectors for in situ delivery of nanobodies against NetB and α toxin, two key toxins associated with NE pathophysiology. We generated nanobodies and showed that these nanobodies neutralize NetB and α toxin. We selected L. reuteri vector strains with intrinsic benefits and demonstrated that these strains inhibit C. perfringens and secrete over 130 metabolites, some of which play a key role in maintaining gut health. Recombinant L. reuteri strains efficiently secreted nanobodies and these nanobodies neutralized NetB. The recombinant strains were genetically and phenotypically stable over 480 generations and showed persistent colonization in chickens. A two-dose in ovo and drinking water administration of recombinant L. reuteri strains protected chickens from NE-associated mortality. These results provide proof-of-concept data for using L. reuteri as a live vector for delivery of nanobodies with broad applicability to other targets and highlight the potential synergistic effects of vector strains and nanobodies for addressing complex diseases such as NE.
28 Dec 2021Submitted to MicrobiologyOpen
29 Dec 2021Submission Checks Completed
29 Dec 2021Assigned to Editor
30 Dec 2021Reviewer(s) Assigned
19 Jan 2022Review(s) Completed, Editorial Evaluation Pending
19 Jan 2022Editorial Decision: Revise Minor
27 Jan 20221st Revision Received
28 Jan 2022Submission Checks Completed
28 Jan 2022Assigned to Editor
28 Jan 2022Review(s) Completed, Editorial Evaluation Pending
28 Jan 2022Reviewer(s) Assigned
07 Feb 2022Editorial Decision: Revise Minor
07 Feb 20222nd Revision Received
10 Feb 2022Submission Checks Completed
10 Feb 2022Assigned to Editor
10 Feb 2022Review(s) Completed, Editorial Evaluation Pending
16 Feb 2022Editorial Decision: Accept