{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T04:32:42Z","timestamp":1776918762587,"version":"3.51.2"},"reference-count":61,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,1,28]],"date-time":"2021-01-28T00:00:00Z","timestamp":1611792000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>Self-amplifying replicon RNA (RepRNA) promotes expansion of mRNA templates encoding genes of interest through their replicative nature, thus providing increased antigen payloads. RepRNA derived from the non-cytopathogenic classical swine fever virus (CSFV) targets monocytes and dendritic cells (DCs), potentially promoting prolonged antigen expression in the DCs, contrasting with cytopathogenic RepRNA. We engineered pestivirus RepRNA constructs encoding influenza virus H5N1 (A\/chicken\/Yamaguchi\/7\/2004) nucleoprotein (Rep-NP) or hemagglutinin (Rep-HA). The inherent RNase-sensitivity of RepRNA had to be circumvented to ensure efficient delivery to DCs for intracellular release and RepRNA translation; we have reported how only particular synthetic delivery vehicle formulations are appropriate. The question remained concerning RepRNA packaged in virus replicon particles (VRPs); we have now compared an efficient polyethylenimine (PEI)-based formulation (polyplex) with VRP-delivery as well as naked RepRNA co-administered with the potent bis-(3\u2019,5\u2019)-cyclic dimeric adenosine monophosphate (c-di-AMP) adjuvant. All formulations contained a Rep-HA\/Rep-NP mix, to assess the breadth of both humoral and cell-mediated defences against the influenza virus antigens. Assessment employed pigs for their close immunological relationship to humans, and as natural hosts for influenza virus. Animals receiving the VRPs, as well as PEI-delivered RepRNA, displayed strong humoral and cellular responses against both HA and NP, but with VRPs proving to be more efficacious. In contrast, naked RepRNA plus c-di-AMP could induce only low-level immune responses, in one out of five pigs. In conclusion, RepRNA encoding different influenza virus antigens are efficacious for inducing both humoral and cellular immune defences in pigs. Comparisons showed that packaging within VRP remains the most efficacious for delivery leading to induction of immune defences; however, this technology necessitates employment of expensive complementing cell cultures, and VRPs do not target human cells. Therefore, choosing the appropriate synthetic delivery vehicle still offers potential for rapid vaccine design, particularly in the context of the current coronavirus pandemic.<\/jats:p>","DOI":"10.3389\/fimmu.2020.622385","type":"journal-article","created":{"date-parts":[[2021,1,28]],"date-time":"2021-01-28T17:39:56Z","timestamp":1611855596000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":16,"title":["Self-Amplifying Pestivirus Replicon RNA Encoding Influenza Virus Nucleoprotein and Hemagglutinin Promote Humoral and Cellular Immune Responses in Pigs"],"prefix":"10.3389","volume":"11","author":[{"given":"Thomas","family":"D\u00e9moulins","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Ruggli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Markus","family":"Gerber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa J.","family":"Thomann-Harwood","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Ebensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kai","family":"Schulze","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos A.","family":"Guzm\u00e1n","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kenneth C.","family":"McCullough","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,1,28]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.0803467","article-title":"Vaccinia virus-based multivalent H5N1 avian influenza vaccines adjuvanted with IL-15 confer sterile cross-clade protection in mice","volume":"182","author":"Poon","year":"2009","journal-title":"J Immunol"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0013162","article-title":"Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses","volume":"5","author":"Price","year":"2010","journal-title":"PloS One"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1192517","article-title":"Induction of broadly neutralizing H1N1 influenza antibodies by vaccination","volume":"329","author":"Wei","year":"2010","journal-title":"Science"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2010.202","article-title":"A universal influenza A vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge","volume":"18","author":"Zhou","year":"2010","journal-title":"Mol Ther"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2017.11.017","article-title":"Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses","volume":"26","author":"Vogel","year":"2018","journal-title":"Mol Ther"},{"key":"B6","first-page":"WO 2009146867","article-title":"Pestivirus replicons providing an RNA-based viral vector system. PCT\/EP2009\/003892","author":"Tratschin","year":"2008"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1002\/jgm.696","article-title":"Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis","volume":"7","author":"Akinc","year":"2005","journal-title":"J Gene Med"},{"key":"B8","article-title":"Replicon-based vectors of positive strand RNA viruses","volume":"2","author":"Khromykh","year":"2000","journal-title":"Curr Opin Mol Ther"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1586\/14760584.2015.965690","article-title":"Self-replicating alphavirus RNA vaccines","volume":"14","author":"Ljungberg","year":"2015","journal-title":"Expert Rev Vaccines"},{"key":"B10","first-page":"28","article-title":"Alphavirus-based vaccines","volume":"4","author":"Lundstrom","year":"2002","journal-title":"Curr Opin Mol Ther"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.4155\/tde.12.90","article-title":"Functional RNA delivery targeted to dendritic cells by synthetic nanoparticles","volume":"3","author":"McCullough","year":"2012","journal-title":"Ther Delivery"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines2040735","article-title":"Self-amplifying replicon RNA vaccine delivery to dendritic cells by synthetic nanoparticles","volume":"2","author":"McCullough","year":"2014","journal-title":"Vaccines"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1517\/14712598.6.2.135","article-title":"Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications","volume":"6","author":"Pijlman","year":"2006","journal-title":"Expert Opin Biol Ther"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1002\/rmv.360","article-title":"Alphavirus vectors and vaccination","volume":"12","author":"Rayner","year":"2002","journal-title":"Rev Med Virol"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a007401","article-title":"Dendritic cells: arbiters of immunity and immunological tolerance","volume":"4","author":"Lewis","year":"2012","journal-title":"Cold Spring Harb Perspect Biol"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2010.10.007","article-title":"Designing vaccines based on biology of human dendritic cell subsets","volume":"33","author":"Palucka","year":"2010","journal-title":"Immunity"},{"key":"B17","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/j.coi.2007.12.002","article-title":"Endocytosis mechanisms and the cell biology of antigen presentation","volume":"20","author":"Burgdorf","year":"2008","journal-title":"Curr Opin Immunol"},{"key":"B18","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1111\/j.0105-2896.2004.00142.x","article-title":"Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens","volume":"199","author":"Heath","year":"2004","journal-title":"Immunol Rev"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1038\/nri2368","article-title":"The known unknowns of antigen processing and presentation","volume":"8","author":"Vyas","year":"2008","journal-title":"Nat Rev Immunol"},{"key":"B20","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/bs.aivir.2015.03.002","article-title":"The Molecular Biology of Pestiviruses","volume":"93","author":"Tautz","year":"2015","journal-title":"Adv Virus Res"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0307145101","article-title":"Mimicking live flavivirus immunization with a noninfectious RNA vaccine","volume":"101","author":"Kofler","year":"2004","journal-title":"Proc Natl Acad Sci USA"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3892\/mmr_00000168","article-title":"Electroporation of RNA stimulates immunity to an encoded reporter gene in mice","volume":"2","author":"Piggott","year":"2009","journal-title":"Mol Med Rep"},{"key":"B23","doi-asserted-by":"publisher","first-page":"e173","DOI":"10.1038\/mtna.2014.24","article-title":"Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo","volume":"3","author":"McCullough","year":"2014","journal-title":"Mol Ther Nucleic Acids"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2017.09.018","article-title":"Self-replicating RNA vaccine functionality modulated by fine-tuning of polyplex delivery vehicle structure","volume":"266","author":"D\u00e9moulins","year":"2017","journal-title":"J Control Release"},{"key":"B25","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1007\/978-1-4939-6481-9_3","article-title":"Self-Replicating RNA Vaccine Delivery to Dendritic Cells","volume":"1499","author":"D\u00e9moulins","year":"2017","journal-title":"Methods Mol Biol"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1016\/j.nano.2015.11.001","article-title":"Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines","volume":"12","author":"D\u00e9moulins","year":"2016","journal-title":"Nanomedicine"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2018.04.019","article-title":"Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic Lipids","volume":"12","author":"Englezou","year":"2018","journal-title":"Mol Ther Nucleic Acids"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0029732","article-title":"Intradermal electroporation of naked replicon RNA elicits strong immune responses","volume":"7","author":"Johansson","year":"2012","journal-title":"PloS One"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.01223","article-title":"Mucosal Administration of Cycle-Di-Nucleotide-Adjuvanted Virosomes Efficiently Induces Protection against Influenza H5N1 in Mice","volume":"8","author":"Ebensen","year":"2017","journal-title":"Front Immunol"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2011.05.026","article-title":"Bis-(3\u2019,5\u2019)-cyclic dimeric adenosine monophosphate: strong Th1\/Th2\/Th17 promoting mucosal adjuvant","volume":"29","author":"Ebensen","year":"2011","journal-title":"Vaccine"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2011.04.094","article-title":"Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Th1 CD4+ cells and strong mucosal and systemic antibody responses in mice","volume":"29","author":"Madhun","year":"2011","journal-title":"Vaccine"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0026973","article-title":"Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP","volume":"6","author":"Pedersen","year":"2011","journal-title":"PloS One"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2018.00505","article-title":"Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression","volume":"9","author":"Vashist","year":"2018","journal-title":"Front Immunol"},{"key":"B34","doi-asserted-by":"publisher","first-page":"287","DOI":"10.1007\/s00441-020-03206-9","article-title":"The pig as a model for immunology research","volume":"380","author":"Pabst","year":"2020","journal-title":"Cell Tissue Res"},{"key":"B35","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1016\/S0034-5288(18)34087-6","article-title":"Establishment, viral susceptibility and biological characteristics of a swine kidney cell line SK-6","volume":"13","author":"Kasza","year":"1972","journal-title":"Res Vet Sci"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.70.6.3478-3487.1996","article-title":"Nucleotide sequence of classical swine fever virus strain Alfort\/187 and transcription of infectious RNA from stably cloned full-length cDNA","volume":"70","author":"Ruggli","year":"1996","journal-title":"J Virol"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-005-0723-6","article-title":"Pathogenicity of a highly pathogenic avian influenza virus, A\/chicken\/Yamaguchi\/7\/04 (H5N1) in different species of birds and mammals","volume":"151","author":"Isoda","year":"2006","journal-title":"Arch Virol"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1051\/vetres:2006028","article-title":"Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application","volume":"37","author":"Frey","year":"2006","journal-title":"Vet Res"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2010.12.026","article-title":"Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-alpha\/beta and carry foreign genes","volume":"29","author":"Suter","year":"2011","journal-title":"Vaccine"},{"key":"B40","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.jviromet.2011.06.007","article-title":"Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation","volume":"176","author":"Eymann-Hani","year":"2011","journal-title":"J Virol Methods"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1016\/j.nano.2013.01.002","article-title":"Alginate-coated chitosan nanogel capacity to modulate the effect of TLR ligands on blood dendritic cells","volume":"9","author":"D\u00e9moulins","year":"2013","journal-title":"Nanomedicine"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1007\/BF01702592","article-title":"Baculovirus expression and affinity purification of protein E2 of classical swine fever virus strain Alfort\/187","volume":"10","author":"Ruggli","year":"1995","journal-title":"Virus Genes"},{"key":"B43","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1051\/vetres:2008030","article-title":"Membrane markers of the immune cells in swine: an update","volume":"39","author":"Piriou-Guzylack","year":"2008","journal-title":"Vet Res"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2003.08.006","article-title":"Attenuation of classical swine fever virus by deletion of the viral N(pro) gene","volume":"22","author":"Mayer","year":"2004","journal-title":"Vaccine"},{"key":"B45","doi-asserted-by":"publisher","first-page":"16379","DOI":"10.1038\/s41598-017-16419-w","article-title":"Virus replicon particle vaccines expressing nucleoprotein of influenza A virus mediate enhanced inflammatory responses in pigs","volume":"7","author":"Ricklin","year":"2017","journal-title":"Sci Rep"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2020.108621","article-title":"The R614E mutation of mouse Mx1 protein contributes to the novel antiviral activity against classical swine fever virus","volume":"243","author":"Chen","year":"2020","journal-title":"Vet Microbiol"},{"key":"B47","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1080\/14712598.2018.1391786","article-title":"Influenza vaccine response: future perspectives","volume":"18","author":"Mameli","year":"2018","journal-title":"Expert Opin Biol Ther"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00237","article-title":"Development of cross-protective influenza a vaccines based on cellular responses","volume":"6","author":"Soema","year":"2015","journal-title":"Front Immunol"},{"key":"B49","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/vaccines8030464","article-title":"Next Generation Influenza Vaccines: Looking into the Crystal Ball","volume":"8","author":"Guzman","year":"2020","journal-title":"Vaccines (Basel)"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02032-06","article-title":"Classical swine fever virus Npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation","volume":"81","author":"Bauhofer","year":"2007","journal-title":"J Virol"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1096\/fj.201601093R","article-title":"Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING","volume":"31","author":"Rueckert","year":"2017","journal-title":"FASEB J"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1086\/588431","article-title":"Plasmid DNA-based vaccines protect mice and ferrets against lethal challenge with A\/Vietnam\/1203\/04 (H5N1) influenza virus","volume":"197","author":"Lalor","year":"2008","journal-title":"J Infect Dis"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0009812","article-title":"Comparative efficacy of hemagglutinin, nucleoprotein, and matrix 2 protein gene-based vaccination against H5N1 influenza in mouse and ferret","volume":"5","author":"Rao","year":"2010","journal-title":"PloS One"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-019-0056-x","article-title":"Next-generation influenza vaccines: opportunities and challenges","volume":"19","author":"Wei","year":"2020","journal-title":"Nat Rev Drug Discovery"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2016.00010","article-title":"The Role of CD4 T Cell Memory in Generating Protective Immunity to Novel and Potentially Pandemic Strains of Influenza","volume":"7","author":"DiPiazza","year":"2016","journal-title":"Front Immunol"},{"key":"B56","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2612","article-title":"Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans","volume":"18","author":"Wilkinson","year":"2012","journal-title":"Nat Med"},{"key":"B57","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2016.10.040","article-title":"Influenza Vaccine Research funded by the European Commission FP7-Health-2013-Innovation-1 project","volume":"34","author":"Liu","year":"2016","journal-title":"Vaccine"},{"key":"B58","article-title":"Functional and phenotypic analysis of porcine peripheral blood CD4\/CD8 double-positive T cells","volume":"87","author":"Zuckermann","year":"1996","journal-title":"Immunology"},{"key":"B59","doi-asserted-by":"publisher","DOI":"10.1006\/cimm.1996.0078","article-title":"Functional characterization of porcine CD4+CD8+ extrathymic T lymphocytes","volume":"168","author":"Summerfield","year":"1996","journal-title":"Cell Immunol"},{"key":"B60","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02133-14","article-title":"Induction of broadly reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans","volume":"88","author":"Nachbagauer","year":"2014","journal-title":"J Virol"},{"key":"B61","doi-asserted-by":"publisher","first-page":"e02556","DOI":"10.1128\/mBio.02556-14","article-title":"Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice","volume":"6","author":"Wohlbold","year":"2015","journal-title":"MBio"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2020.622385\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,1,28]],"date-time":"2021-01-28T17:39:58Z","timestamp":1611855598000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2020.622385\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,28]]},"references-count":61,"alternative-id":["10.3389\/fimmu.2020.622385"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2020.622385","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,28]]},"article-number":"622385"}}