Surface engineered polyanhydride-based oral Salmonella subunit nanovaccine for poultry

Sankar Renu1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA, [email protected];2 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA, [email protected]

Ashley D Markazi3 Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA

Santosh Dhakal1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA, [email protected];2 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA, [email protected]

Yashavanth S Lakshmanappa1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA, [email protected];2 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA, [email protected]

Suren R Gourapura1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA, [email protected];2 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA, [email protected]

Revathi Shanmugasundaram3 Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA

Sujata Senapati4 Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA

Balaji Narasimhan4 Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA

Ramesh K Selvaraj5 Department of Poultry Science, University of Georgia, Athens, GA 30602, USA, [email protected]Correspondence[email protected]

Gourapura J Renukaradhya1 Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA, [email protected];2 Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA, [email protected]Correspondence[email protected]

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Abstract

Purpose

Salmonellosis is a severe economic threat in poultry and a public health concern. Currently available vaccines are ineffective, and thus, developing effective oral Salmonella vaccine is warranted. Especially, a potent oral vaccine such as the mucoadhesive polyanhydride nanoparticle (PNP) protects the vaccine cargo and delivers to intestinal immune sites to elicit robust mucosal immunity and mitigate Salmonella colonization and shedding.

Materials and methods

We designed a Salmonella subunit vaccine using PNP containing immunogenic Salmonella outer membrane proteins (OMPs) and flagellar (F) protein-entrapped and surface F-protein-coated PNPs (OMPs-F-PNPs) using a solvent displacement method. Using high-throughput techniques, we characterized the OMPs-F-PNPs physicochemical properties and analyzed its efficacy in layer birds vaccinated orally.

Results

The candidate vaccine was resistant in acidic microenvironment and had ideal physicochemical properties for oral delivery in terms of particle size, charge, morphology, biocompatibility, and pH stability. In vitro, in vivo, and ex vivo studies showed that F-protein surface-anchored nanoparticles were better targeted to chicken immune cells in peripheral blood and splenocytes and intestinal Peyer’s patch sites. In layer chickens inoculated orally with OMPs-F-PNPs, substantially higher OMPs-specific IgG response and secretion of Th1 cytokine IFN-γ in the serum, enhanced CD8⁺/CD4⁺ cell ratio in spleen, and increased OMPs-specific lymphocyte proliferation were observed. OMPs-F-PNPs vaccination also upregulated the expression of toll-like receptor (TLR)-2 and -4, TGF-β, and IL-4 cytokines’ genes in chicken cecal tonsils (lymphoid tissues). Importantly, OMPs-F-PNPs vaccine cleared Salmonella cecal colonization in 33% of vaccinated birds.

Conclusion

This pilot in vivo study demonstrated the targeted delivery of OMPs-F-PNPs to ileum mucosal immune sites of chickens and induced specific immune response to mitigate Salmonella colonization in intestines.

Keywords:

Supplementary materials

Figure S1 SDS-PAGE analyses of isolated Salmonella protein extracts.

Notes: Lane 1: standard protein marker (kDa); Lane 2: F-protein; and Lane 3: OMPs.

Abbreviations: F, flagellar; OMPs, outer membrane proteins.

Figure S2 Analysis of biocompatibility and pH stability of PNPs.

Notes: (A) Hemolysis assay using chicken RBCs treated with (1) Triton X-100; (2) PBS; and (3–6) various indicated concentrations of PNPs. (B) Simple microscopy pictures of chicken RBCs incubated with (a) Triton X-100; (b) PBS; and (c) PNPs (1,000 µg/mL) for 3 hours. (C) In vitro pH stability of PNPs treated for 1, 2, and 3 hours.

Abbreviations: PNPs, polyanhydride nanoparticles; RBCs, red blood cells.

Figure S2 Analysis of biocompatibility and pH stability of PNPs.Notes: (A) Hemolysis assay using chicken RBCs treated with (1) Triton X-100; (2) PBS; and (3–6) various indicated concentrations of PNPs. (B) Simple microscopy pictures of chicken RBCs incubated with (a) Triton X-100; (b) PBS; and (c) PNPs (1,000 µg/mL) for 3 hours. (C) In vitro pH stability of PNPs treated for 1, 2, and 3 hours.Abbreviations: PNPs, polyanhydride nanoparticles; RBCs, red blood cells.

Figure S3 Mucoadhesive nature of fluorescent-tagged PNPs-F in the chicken ileum by ex vivo analysis.

Notes: Ileum was harvested from healthy layer chickens and treated with PBS, RITC dye, and RITC dye-tagged PNPs-F in PBS for 4 hours, washed, fixed, sectioned, stained with DAPI, and examined under a fluorescent microscope. The images were obtained at 2× magnification, and scale bar is 2 mm. PNPs-F, surface F-protein-coated PNPs.

Abbreviations: PNPs, polyanhydride nanoparticles; RITC, rhodamine B isothiocyanate.

Figure S3 Mucoadhesive nature of fluorescent-tagged PNPs-F in the chicken ileum by ex vivo analysis.Notes: Ileum was harvested from healthy layer chickens and treated with PBS, RITC dye, and RITC dye-tagged PNPs-F in PBS for 4 hours, washed, fixed, sectioned, stained with DAPI, and examined under a fluorescent microscope. The images were obtained at 2× magnification, and scale bar is 2 mm. PNPs-F, surface F-protein-coated PNPs.Abbreviations: PNPs, polyanhydride nanoparticles; RITC, rhodamine B isothiocyanate.

Figure S4 Flow cytometry gating pattern of control, IgG isotype, CD4⁺, CD8⁺, and CD4⁺CD8⁺ cells.

Figure S5 Detection of live S. enteritidis in the ceca of chickens orally inoculated with OMPs-F-PNPs and Salmonella-challenged.

Notes: Fresh cecal samples were tested for live S. enteritidis by culture method. Initially, bacteria were enriched in peptone water (2× concentration) for 12 hours at 37°C, followed by streaking on the XLT plates. Representative colonies from the plates were confirmed as Salmonella by standard methods. Each bar represents the S. enteritidis-positive/negative chickens under each group. OMPs-F-PNPs, OMPs and F-protein-entrapped and surface F-protein-coated PNPs.

Abbreviations: Ch, challenge; F, flagellar; OMPs, outer membrane proteins; PNPs, polyanhydride nanoparticles; S. enteritidis, Salmonella enteritidis; XLT, xylose lysine tergitol.

Figure S5 Detection of live S. enteritidis in the ceca of chickens orally inoculated with OMPs-F-PNPs and Salmonella-challenged.Notes: Fresh cecal samples were tested for live S. enteritidis by culture method. Initially, bacteria were enriched in peptone water (2× concentration) for 12 hours at 37°C, followed by streaking on the XLT plates. Representative colonies from the plates were confirmed as Salmonella by standard methods. Each bar represents the S. enteritidis-positive/negative chickens under each group. OMPs-F-PNPs, OMPs and F-protein-entrapped and surface F-protein-coated PNPs.Abbreviations: Ch, challenge; F, flagellar; OMPs, outer membrane proteins; PNPs, polyanhydride nanoparticles; S. enteritidis, Salmonella enteritidis; XLT, xylose lysine tergitol.

Acknowledgments

We thank our animal care unit staff Mr Keith Patterson and Jack Sidle for help in chicken studies. Dr Tea Meulia and Leona Horst and her team at MCIC, OARDC, provided help in FESEM analysis. We sincerely thank Dr Steven Krakowka for his help in editing the manuscript. This work was partially supported by the USDA-NIFA grant and OSU Accelerator Award. Salaries and research support were provided by state and federal funds appropriated to OARDC, The Ohio State University.

Disclosure

The authors report no conflicts of interest in this work.