Construction of a Lactobacillus plantarum Strain Expressing the Capsid Protein of Porcine Circovirus Type 2d (PCV2d) as an Oral Vaccine

Abstract

Porcine circovirus type 2 (PCV2) is a pathogenic virus that causes high rates of porcine death, resulting in severe economic losses to the swine industry. In recent years, the prevalence of PCV2d genotype infection in pigs has increased, but most commercially available vaccines were developed against the PCV2a strain and do not ensure complete protection from PCV2d. Here, we first constructed an expression vector for the antigenic ORF2-encoded capsid protein of PCV2d (pLp3050-His6-tag-capsid). We then utilized Lactobacillus plantarum to express the protein at mucosal sites in orally vaccinated mice. After transducing L. plantarum with pLp3050-His6-tag-capsid, the expressed protein could be found in cell wall and cell-free supernatant fractions by Western blotting. Using flow cytometry, we found that L. plantarum cells with surface-displayed capsid protein increased with time after SppIP induction. Finally, mice that were orally immunized 18 times with capsid-expressing L. plantarum showed increased levels of capsid-specific sIgA and virus neutralizing activity at mucosal sites, suggesting mucosal immunity had been stimulated by the vaccine. Overall, our findings demonstrate the feasibility and utility of a PCV2d-based vaccine, which may be of great value in porcine agriculture.

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References

  1. 1.

    Chae C (2005) A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169:326–336. https://doi.org/10.1016/j.tvjl.2004.01.012

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Meng XJ (2012) Emerging and re-emerging swine viruses. Transbound Emerg Dis 59:85–102. https://doi.org/10.1111/j.1865-1682.2011.01291.x

    Article  PubMed  Google Scholar 

  3. 3.

    Nawagitgul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS (2000) Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81:2281–2287. https://doi.org/10.1099/0022-1317-81-9-2281

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Choi CY, Choi YC, Park IB, Lee CH, Kang SJ, Chun T (2018) The ORF5 protein of porcine circovirus type 2 enhances viral replication by dampening type I interferon expression in porcine epithelial cells. Vet Microbiol 226:50–58. https://doi.org/10.1016/j.vetmic.2018.10.005

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    He J, Cao J, Zhou N, Jin Y, Wu J, Zhou J (2013) Identification and functional analysis of the novel ORF4 protein encoded by porcine circovirus type 2. J Virol 87:1420–1429. https://doi.org/10.1128/JVI.01443-12

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Khayat R, Brunn N, Speir JA, Hardham JM, Ankenbauer RG, Schneemann A, Johnson JE (2011) The 2.3-angstrom structure of porcine circovirus 2. J Virol 85:7856–7862. https://doi.org/10.1128/jvi.00737-11

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Xiao CT, Halbur PG, Opriessnig T (2015) Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d. J Gen Virol 96:1830–1841. https://doi.org/10.1099/vir.0.000100

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Franzo G, Cortey M, de Castro AM, Piovezan U, Szabo MP, Drigo M, Segales J, Richtzenhain LJ (2015) Genetic characterisation of Porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: an opportunity to reconstruct the history of PCV2 evolution. Vet Microbiol 178:158–162. https://doi.org/10.1016/j.vetmic.2015.05.003

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Olvera A, Cortey M, Segales J (2007) Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality. Virology 357:175–185. https://doi.org/10.1016/j.virol.2006.07.047

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Xiao C-T, Harmon KM, Halbur PG, Opriessnig T (2016) PCV2d-2 is the predominant type of PCV2 DNA in pig samples collected in the US during 2014–2016. Vet Microbiol 197:72–77

    CAS  Article  Google Scholar 

  11. 11.

    Yao J, Qin YR, Zeng Y, Ouyang K, Chen Y, Huang WJ, Wei ZZ (2019) Genetic analysis of porcine circovirus type 2 (PCV2) strains between 2002 and 2016 reveals PCV2 mutant predominating in porcine population in Guangxi, China. Bmc Vet Res. https://doi.org/10.1186/s12917-019-1859-z

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Yang S, Yin S, Shang Y, Liu B, Yuan L, Khan MUZ, Liu X, Cai J (2018) Phylogenetic and genetic variation analyses of porcine circovirus type 2 isolated from China. Transbound Emerg Dis 65:e383–e392. https://doi.org/10.1111/tbed.12768

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Palya V, Homonnay ZG, Mato T, Kiss I (2018) Characterization of a PCV2d-2 isolate by experimental infection of pigs. Virol J 15:185. https://doi.org/10.1186/s12985-018-1098-0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Fenaux M, Opriessnig T, Halbur PG, Meng XJ (2003) Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs. J Virol 77:11232–11243

    CAS  Article  Google Scholar 

  15. 15.

    Opriessnig T, Meng XJ, Halbur PG (2007) Porcine circovirus type 2-associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19:591–615. https://doi.org/10.1177/104063870701900601

    Article  PubMed  Google Scholar 

  16. 16.

    Opriessnig T, Gerber PF, Xiao C-T, Mogler M, Halbur PG (2014) A commercial vaccine based on PCV2a and an experimental vaccine based on a variant mPCV2b are both effective in protecting pigs against challenge with a 2013 US variant mPCV2b strain. Vaccine 32:230–237

    CAS  Article  Google Scholar 

  17. 17.

    Opriessnig T, Xiao CT, Halbur PG, Gerber PF, Matzinger SR, Meng XJ (2017) A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naive pigs under experimental conditions. Vaccine 35:248–254. https://doi.org/10.1016/j.vaccine.2016.11.085

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Lekcharoensuk P, Morozov I, Paul PS, Thangthumniyom N, Wajjawalku W, Meng XJ (2004) Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2. J Virol 78:8135–8145. https://doi.org/10.1128/Jvi.78.15.8135-8145.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Guo LJ, Fu YJ, Wang YP, Lu YH, Wei YW, Tang QH, Fan PH, Liu JB, Zhang L, Zhang FY, Huang LP, Liu D, Li SB, Wu HL, Liu CM (2012) A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain. Plos One. https://doi.org/10.1371/journal.pone.0041463

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Khangwal I, Shukla P (2019) Potential prebiotics and their transmission mechanisms: recent approaches. J Food Drug Anal 27:649–656. https://doi.org/10.1016/j.jfda.2019.02.003

    Article  PubMed  Google Scholar 

  21. 21.

    Khangwal I, Shukla P (2019) Prospecting prebiotics, innovative evaluation methods, and their health applications: a review. 3 Biotech. https://doi.org/10.1007/s13205-019-1716-6

    Article  PubMed  Google Scholar 

  22. 22.

    Barcelos MCS, Vespermann KAC, Pelissari FM, Molina G (2019) Current status of biotechnological production and applications of microbial exopolysaccharides. Crit Rev Food Sci Nutr. https://doi.org/10.1080/10408398.2019.1575791

    Article  PubMed  Google Scholar 

  23. 23.

    Castro-Bravo N, Margolles A, Wells JM, Ruas-Madiedo P (2019) Exopolysaccharides synthesized by Bifidobacterium animalis subsp. lactis interact with TLR4 in intestinal epithelial cells. Anaerobe 56:98–101. https://doi.org/10.1016/j.anaerobe.2019.02.014

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Yadav R, Singh PK, Shukla P (2018) Metabolic engineering for probiotics and their genome-wide expression profiling. Curr Protein Pept Sci 19:68–74. https://doi.org/10.2174/1389203718666161111130157

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Yadav R, Shukla P (2017) An overview of advanced technologies for selection of probiotics and their expediency: a review. Crit Rev Food Sci 57:3233–3242. https://doi.org/10.1080/10408398.2015.1108957

    CAS  Article  Google Scholar 

  26. 26.

    Chowdhury MY, Li R, Kim JH, Park ME, Kim TH, Pathinayake P, Weeratunga P, Song MK, Son HY, Hong SP, Sung MH, Lee JS, Kim CJ (2014) Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice. PLoS ONE 9:e94051. https://doi.org/10.1371/journal.pone.0094051

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Kuczkowska K, Kleiveland CR, Minic R, Moen LF, Overland L, Tjaland R, Carlsen H, Lea T, Mathiesen G, Eijsink VG (2017) Immunogenic properties of Lactobacillus plantarum producing surface-displayed mycobacterium tuberculosis antigens. Appl Environ Microbiol. https://doi.org/10.1128/aem.02782-16

    Article  PubMed  Google Scholar 

  28. 28.

    Kajikawa A, Zhang L, LaVoy A, Bumgardner S, Klaenhammer TR, Dean GA (2015) Mucosal immunogenicity of genetically modified Lactobacillus acidophilus expressing an HIV-1 epitope within the surface layer protein. Plos One. https://doi.org/10.1371/journal.pone.0141713

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Yadav R, Singh PK, Puniya AK, Shukla P (2016) Catalytic interactions and molecular docking of bile salt hydrolase (BSH) from L. plantarum RYPR1 and its prebiotic utilization. Front Microbiol 7:2116. https://doi.org/10.3389/fmicb.2016.02116

    Article  PubMed  Google Scholar 

  30. 30.

    Dahiya DK, Renuka Puniya M, Shandilya UK, Dhewa T, Kumar N, Kumar S, Puniya AK, Shukla P (2017) Gut microbiota modulation and its relationship with obesity using prebiotic fibers and probiotics: a review. Front Microbiol. https://doi.org/10.3389/fmicb.2017.00563

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Liu YW, Su YW, Ong WK, Cheng TH, Tsai YC (2011) Oral administration of Lactobacillus plantarum K68 ameliorates DSS-induced ulcerative colitis in BALB/c mice via the anti-inflammatory and immunomodulatory activities. Int Immunopharmacol 11(12):2159–2166. https://doi.org/10.1016/j.intimp.2011.09.013

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Halbmayr E, Mathiesen G, Nguyen T-H, Maischberger T, Peterbauer CK, Eijsink VG, Haltrich D (2008) High-level expression of recombinant β-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a sakacin P-based expression system. J Agric Food Chem 56:4710–4719

    CAS  Article  Google Scholar 

  33. 33.

    Aukrust TW, Brurberg MB, Nes IF (1995) Transformation of Lactobacillus by electroporation. Methods Mol Biol 47:201–208. https://doi.org/10.1385/0-89603-310-4:201

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Piard JC, Hautefort I, Fischetti VA, Ehrlich SD, Fons M, Gruss A (1997) Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J Bacteriol 179:3068–3072. https://doi.org/10.1128/jb.179.9.3068-3072.1997

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG (2009) Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genom 10:425

    Article  Google Scholar 

  36. 36.

    Mathiesen G, Sveen A, Piard JC, Axelsson L, Eijsink VGH (2008) Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides. J Appl Microbiol 105:215–226. https://doi.org/10.1111/j.1365-2672.2008.03734.x

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Michon C, Kuczkowska K, Langella P, Eijsink VG, Mathiesen G, Chatel JM (2015) Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo. Microb Cell Fact 14:95. https://doi.org/10.1186/s12934-015-0290-9

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Brandtzaeg P (1988) Immunobarriers of the mucosa of the upper respiratory and digestive pathways. Acta Otolaryngol 105:172–180

    CAS  Article  Google Scholar 

  39. 39.

    Aziz MA, Midha S, Waheed SM, Bhatnagar R (2007) Oral vaccines: new needs, new possibilities. BioEssays 29:591–604. https://doi.org/10.1002/bies.20580

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Sasikumar P, Gomathi S, Anbazhagan K, Selvam GS (2013) Secretion of biologically active heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 using homologous signal peptides. BioMed Res Int 2013:280432. https://doi.org/10.1155/2013/280432

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Wang K, Huang L, Kong J, Zhang X (2008) Expression of the capsid protein of porcine circovirus type 2 in Lactococcus lactis for oral vaccination. J Virol Methods 150:1–6. https://doi.org/10.1016/j.jviromet.2008.02.014

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Vesa T, Pochart P, Marteau P (2000) Pharmacokinetics of Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363 in the human gastrointestinal tract. Aliment Pharm Therap 14:823–828

    CAS  Article  Google Scholar 

  43. 43.

    Michon C, Langella P, Eijsink VGH, Mathiesen G, Chatel JM (2016) Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications. Microb Cell Fact. https://doi.org/10.1186/s12934-016-0468-9

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Antelmann H, Darmon E, Noone D, Veening JW, Westers H, Bron S, Kuipers OP, Devine KM, Hecker M, van Dijl JM (2003) The extracellular proteome of Bacillus subtilis under secretion stress conditions. Mol Microbiol 49:143–156

    CAS  Article  Google Scholar 

  45. 45.

    Bolhuis A, Tjalsma H, Smith HE, de Jong A, Meima R, Venema G, Bron S, van Dijl JM (1999) Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis. Appl Environ Microbiol 65:2934–2941

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Fort M, Olvera A, Sibila M, Segales J, Mateu E (2007) Detection of neutralizing antibodies in postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs. Vet Microbiol 125:244–255. https://doi.org/10.1016/j.vetmic.2007.06.004

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Bermudez-Humaran LG, Cortes-Perez NG, Le Loir Y, Alcocer-Gonzalez JM, Tamez-Guerra RS, de Oca-Luna RM, Langella P (2004) An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci. J Med Microbiol 53:427–433. https://doi.org/10.1099/jmm.0.05472-0

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Wells JM, Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6:349–362. https://doi.org/10.1038/nrmicro1840

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Kuczkowska K, Kleiveland CR, Minic R, Moen LF, Overland L, Tjaland R, Carlsen H, Lea T, Mathiesen G, Eijsink VGH (2017) Immunogenic properties of Lactobacillus plantarum producing surface-displayed mycobacterium tuberculosis antigens. Appl Environ Microbiol. https://doi.org/10.1128/aem.02782-16

    Article  PubMed  Google Scholar 

  50. 50.

    Grassly NC, Kang G, Kampmann B (2015) Biological challenges to effective vaccines in the developing world. Philos Trans R Soc Lond B Biol Sci 370:1671. https://doi.org/10.1098/rstb.2014.0138

    Article  Google Scholar 

  51. 51.

    Neutra MR, Kozlowski PA (2006) Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6:148–158. https://doi.org/10.1038/nri1777

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Ding C, Ma J, Dong Q, Liu Q (2018) Live bacterial vaccine vector and delivery strategies of heterologous antigen: a review. Immunol Lett 197:70–77. https://doi.org/10.1016/j.imlet.2018.03.006

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank Dr. Geir Mathiesen (Norwegian University of Life Sciences, Akershus, Norway) for kindly providing pLp3050 plasmid. This study was supported by the fund from the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-002-041).

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Correspondence to Shu-Chen Hsieh.

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This animal experiment was approved by the by the Institutional Animal Care and Use Committee of the National Taiwan University (NTU-IACUC/protocol 125/2013). All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted.

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Tseng, YH., Hsieh, CC., Kuo, TY. et al. Construction of a Lactobacillus plantarum Strain Expressing the Capsid Protein of Porcine Circovirus Type 2d (PCV2d) as an Oral Vaccine. Indian J Microbiol 59, 490–499 (2019). https://doi.org/10.1007/s12088-019-00827-9

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Keywords

  • Capsid protein
  • Lactobacillus plantarum
  • Mucosal immunity
  • Porcine circovirus type 2d
  • sIgA