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An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice

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Abstract

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The first report on the recombinant production of a candidate vaccine in the moss system.

Abstract

The need for economical and efficient platforms for vaccine production demands the exploration of emerging host organisms. In this study, the production of an antigenic protein is reported employing the moss Physcomitrella patens as an expression host. A multi-epitope protein from the Human Immunodeficiency Virus (HIV) based on epitopes from gp120 and gp41 was designed as a candidate subunit vaccine and named poly-HIV. Transgenic moss plants were generated carrying the corresponding poly-HIV transgene under a novel moss promoter and subsequently seven positive lines were confirmed by PCR. The poly-HIV protein accumulated up to 3.7 µg g−1 fresh weight in protonema cultures. Antigenic and immunogenic properties of the moss-produced recombinant poly-HIV are evidenced by Western blots and by mice immunization assays. The elicitation of specific antibodies in mice was observed, reflecting the immunogenic potential of this moss-derived HIV antigen. This is the first report on the production of a potential vaccine in the moss system and opens the avenue for glycoengineering approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.

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References

  • AVMA (2013) Guidelines for the euthanasia of animals: 2013 edition. American Veterinary Medical Association 1931 N. Meacham road, Schaumburg, IL 60173. ISBN 978-1-882691-21-0

  • Baur A, Reski R, Gorr G (2005) Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol J 3:331–340

    Article  CAS  PubMed  Google Scholar 

  • Buettner-Mainik A, Parsons J, Jerome H, Hartmann A, Lamer S, Schaaf A, Schlosser A, Zipfel PF, Reski R, Decker EL (2011) Production of biologically active recombinant human factor H in Physcomitrella. Plant Biotechnol J 9:373–383

    Article  CAS  Google Scholar 

  • Cueno ME, Hibi Y, Karamatsu K, Yasutomi Y, Imai K, Laurena AC, Okamoto T (2010) Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant. Transgenic Res 19:889–895

    Article  CAS  PubMed  Google Scholar 

  • De Virgilio M, De Marchis F, Bellucci M, Mainieri D, Rossi M, Benvenuto E, Arcioni S, Vitale A (2008) The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin. J Exp Bot 59:2815–2829

    Article  PubMed Central  PubMed  Google Scholar 

  • Decker EL, Reski R (2007) Moss bioreactors producing improved biopharmaceuticals. Curr Opin Biotechnol 18:393–398

    Article  CAS  PubMed  Google Scholar 

  • Decker EL, Reski R (2008) Current achievements in the production of complex biopharmaceuticals with moss bioreactor. Bioprocess Biosyst Eng 31:3–9

    Article  CAS  PubMed  Google Scholar 

  • Decker EL, Reski R (2012) Glycoprotein production in moss bioreactors. Plant Cell Rep 31:453–460

    Article  CAS  PubMed  Google Scholar 

  • Decker EL, Parsons J, Reski R (2014) Glyco-engineering for biopharmaceutical production in moss bioreactors. Front Plant Sci 5:346

    Article  PubMed Central  PubMed  Google Scholar 

  • Dong XN, Wu Y, Chen Y (2005) The neutralizing epitope ELDKWA on HIV-1 gp41: genetic variability and antigenicity. Immunol Lett 101:81–86

    Article  CAS  PubMed  Google Scholar 

  • Excler JL, Robb ML, Kim JH (2014) HIV-1 vaccines: challenges and new perspectives. Hum Vaccine Immunother 10:1734–1746

    Article  CAS  Google Scholar 

  • Forsell MN, Schief WR, Wyatt RT (2009) Immunogenicity of HIV-1 envelope glycoprotein oligomers. Curr Opin HIV AIDS 4:380–387

    Article  PubMed  Google Scholar 

  • Frank W, Decker EL, Reski R (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7:220–227

    Article  CAS  PubMed  Google Scholar 

  • Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30:733–744

    Article  CAS  PubMed  Google Scholar 

  • Girard MP, Osmanov SK, Kieny MP (2006) A review of vaccine research and development: the human immunodeficiency virus (HIV). Vaccine 24:4062–4081

    Article  CAS  PubMed  Google Scholar 

  • Gitzinger M, Parsons J, Reski R, Fussenegger M (2009) Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnol J 7:73–86

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez-Rabade N, McGowan EG, Zhou F, McCabe M, Bock R, Dix PJ, Gray JC, Ma JKC (2011) Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice. Plant Biotechnol J 9:629–638

    Article  CAS  PubMed  Google Scholar 

  • Govea-Alonso DO, Gomez-Cardona EE, Rubio-Infante N, Garcia-Hernandez AL, Varona-Santos JT, Salgado-Bustamante M, Korban SS, Moreno-Fierros L, Rosales-Mendoza S (2013) Production of an antigenic C4(V3)6 multiepitopic HIV protein in bacterial and plant systems. Plant Cell Tissue Organ Cult 113:73–79

    Article  CAS  Google Scholar 

  • Graham BS, McElrath MJ, Keefer MC, Rybczyk K, Berger D, Weinhold KJ, Ottinger J, Ferrari G, Montefiori DC, Stablein D, Smith C, Ginsberg R, Eldridge J, Duerr A, Fast P, Haynes BF (2010) Immunization with cocktail of HIV-derived peptides in Montanide ISA-51 is immunogenic, but causes sterile abscesses and unacceptable reactogenicity. PLoS One 5:e11995

    Article  PubMed Central  PubMed  Google Scholar 

  • Haynes BF, Ma B, Montefiori DC, Wrin T, Petropoulos CJ, Sutherland LL, Scearce RM, Denton C, Xia SM, Korber BT, Liao HX (2006) Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates. Virology 345:44–55

    Article  CAS  PubMed  Google Scholar 

  • Hohe A, Decker EL, Gorr G, Schween G, Reski R (2002) Tight control of growth and cell differentiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor cultures. Plant Cell Rep 20:1135–1140

    Article  CAS  Google Scholar 

  • Hohe A, Egener T, Lucht JM, Holtorf H, Reinhard C, Schween G, Reski R (2004) An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene knockouts in a moss, Physcomitrella patens. Curr Genet 44:339–347

    Article  CAS  PubMed  Google Scholar 

  • Huether CM, Lienhart O, Baur A, Stemmer C, Gorr G, Reski R, Decker EL (2005) Glyco-engineering of moss lacking plant-specific sugar residues. Plant Biol 7:292–299

    Article  CAS  PubMed  Google Scholar 

  • Kajikawa A, Zhang L, Long J, Nordone S, Stoeker L, LaVoy A, Bumgardner S, Klaenhammer T, Dean G (2012) Construction and immunological evaluation of dual cell surface display of HIV-1 Gag and Salmonella enterica serovar Typhimurium FliC in Lactobacillus acidophilus for vaccine delivery. Clin Vaccine Immunol 19:1374–1381

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Koprivova A, Altmann F, Gorr G, Kopriva S, Reski R, Decker EL (2003) N-glycosylation in the moss Physcomitrella patens is organized similarly to higher plants. Plant Biol 5:582–591

    Article  CAS  Google Scholar 

  • Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, Reski R, Decker EL (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2:517–523

    Article  CAS  PubMed  Google Scholar 

  • Krachmarov CP, Kayman SC, Honnen WJ, Trochev O, Pinter A (2001) V3-specific polyclonal antibodies affinity purified from sera of infected humans effectively neutralize primary isolates of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 17:1737–1748

    Article  CAS  PubMed  Google Scholar 

  • Kubo M, Imai A, Nishiyama T, Ishikawa M, Sato Y, Kurata T, Hiwatashi Y, Reski R, Hasebe M (2013) System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens. PLoS One 8:e77356

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Letvin NL, Robinson S, Rohne D, Axthelm MK, Fanton JW, Bilska M, Palker TJ, Liao HX, Haynes BF, Montefiori DC (2001) Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope. J Virol 75:4165–4175

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Liao HX, Etemad-Moghadam B, Montefiori DC, Sun Y, Sodroski J, Scearce RM, Doms RW, Thomasch JR, Robinson S, Letvin NL, Haynes BF (2000) Induction of antibodies in guinea pigs and rhesus monkeys against the human immunodeficiency virus type 1 envelope: neutralization of nonpathogenic and pathogenic primary isolate simian/human immunodeficiency virus strains. J Virol 74:254–263

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mascola JR, Snyder SW, Weislow OS, Belay SM, Belshe RB, Schwartz DH, Clements ML, Dolin R, Graham BS, Gorse GJ, Keefer MC, McElrath MJ, Walker MC, Wagner KF, McNeil JG, McCutchan FE, Burke DS (1996) Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. J Infect Dis 173:340–348

    Article  CAS  PubMed  Google Scholar 

  • Mueller SJ, Lang D, Hoernstein SNW, Lang EGE, Schuessele C, Schmidt A, Fluck M, Leisibach D, Niegl C, Zimmer AD, Schlosser A, Reski R (2014) Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation. Plant Physiol 164:2081–2095

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Müller K, Siegel D, Rodriguez-Jahnke F, Gerrer K, Wend S, Decker EL, Reski R, Weber W, Zurbriggen MD (2014) A red light-controlled synthetic gene expression switch for plant systems. Mol BioSyst 10:1679–1688

    Article  PubMed  Google Scholar 

  • Parsons J, Altmann F, Arrenberg CK, Koprivova A, Beike AK, Stemmer C, Gorr G, Reski R, Decker EL (2012) Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. Plant Biotechnol J 10:851–861

    Article  CAS  PubMed  Google Scholar 

  • Parsons J, Altmann F, Graf M, Stadlmann J, Reski R, Decker EL (2013) A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin. Sci Rep 3:3019

    Article  PubMed Central  PubMed  Google Scholar 

  • Patterson LJ, Robey F, Muck A, van Remoortere K, Aldrich K, Richardson E, Alvord WG, Markham PD, Cranage M, Robert-Guroff M (2001) A conformational C4 peptide polymer vaccine coupled with live recombinant vector priming is immunogenic but does not protect against rectal SIV challenge. AIDS Res Hum Retroviruses 17:837–849

    Article  CAS  PubMed  Google Scholar 

  • Potter M (1985) History of the BALB/c family. Curr Top Microbiol Immunol 122:1–5

    CAS  PubMed  Google Scholar 

  • Reski R, Abel WO (1985) Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine. Planta 165:354–358

    Article  CAS  PubMed  Google Scholar 

  • Rosales-Mendoza S, Rubio-Infante N, Govea-Alonso DO, Moreno-Fierros L (2012) Current status and perspectives of plant-based candidate vaccines against the human immunodeficiency virus (HIV). Plant Cell Rep 31:495–511

    Article  CAS  PubMed  Google Scholar 

  • Rosales-Mendoza S, Orellana-Escobedo L, Romero-Maldonado A, Decker EL, Reski R (2014a) The potential of Physcomitrella patens as a platform for the production of plant-based vaccines. Expert Rev Vaccines 13:203–212

    Article  CAS  PubMed  Google Scholar 

  • Rosales-Mendoza S, Rubio-Infante N, Monreal-Escalante E, Govea-Alonso DO, García-Hernández AL, Salazar-González JA, González-Ortega O, Paz-Maldonado LMT, Moreno-Fierros L (2014b) Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine. Plant Cell Tissue Organ Cult 116:111–123

    Article  CAS  Google Scholar 

  • Rubio-Infante N, Govea-Alonso DO, Alpuche-Solis AG, Garcia-Hernandez AL, Soria-Guerra RE, Paz-Maldonado LMT, Ilhuicatzi-Alvarado D, Varona-Santos JT, Verdin-Teran L, Korban SS, Moreno-Fierros L, Rosales-Mendoza S (2012) A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice. Plant Mol Biol 78:337–349

    Article  CAS  PubMed  Google Scholar 

  • Schuster M, Jost W, Mudde G, Wiederkum S, Schwager C, Janzek E, Altmann F, Stadlmann J, Stemmer C, Gorr G (2007) In vivo glyco-engineered antibody with lytic potential produced by an innovative non-mammalian expression system. Biotechnol J 2:700–708

    Article  CAS  PubMed  Google Scholar 

  • Schween G, Fleig S, Reski R (2002) High-throughput-PCR screen of 15,000 transgenic Physcomitrella plants. Plant Mol Biol Rep 20:43–47

    Article  CAS  Google Scholar 

  • Stott EJ, Almond N, Kent K, Walker B, Hull R, Rose J, Silvera P, Sangster R, Corcoran T, Lines J, Silvera K, Luciw P, Murphy-Corb M, Momin P, Bruck C (1998) Evaluation of a candidate human immunodeficiency virus type 1 (HIV-1) vaccine in macaques: effect of vaccination with HIV-1 gp120 on subsequent challenge with heterologous simian immunodeficiency virus-HIV-1 chimeric virus. J Gen Virol 79:423–432

    CAS  PubMed  Google Scholar 

  • Weise A, Altmann F, Rodriguez-Franco M, Sjoberg ER, Baumer W, Launhardt H, Kietzmann M, Gorr G (2007) High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Δ-fuc-t Δ-xyl-t mutant. Plant Biotechnol J 5:389–401

    Article  CAS  PubMed  Google Scholar 

  • White HN, Meng Q (2012) Diversification of specificity after maturation of the antibody response to the HIV gp41 epitope ELDKWA. PLoS One 7:e31555

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • WHO (2014) World Health Organization on HIV/AIDS. Fact Sheet No. 360, Geneva, Sep 2014. http://www.who.int/hiv/data/en/

  • Zimmer AD, Lang D, Buchta K, Rombauts S, Nishiyama T, Hasebe M, van de Peer Y, Rensing SA, Reski R (2013) Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions. BMC Genom 14:498

    Article  CAS  Google Scholar 

  • Zolla-Pazner S, Cohen SS, Krachmarov C, Wang S, Pinter A, Lu S (2008) Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope. Virology 372:233–246

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Excellence Initiative of the German Federal and State Governments (EXC294 to R.R.). L.O.-E and S.R.-M received DAAD fellowships to conduct part of this project. We are grateful to Anne Katrin Prowse for proofreading of the manuscript.

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The authors declare no conflict of interests.

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Correspondence to Ralf Reski.

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Communicated by Stefan Schillberg.

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Orellana-Escobedo, L., Rosales-Mendoza, S., Romero-Maldonado, A. et al. An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice. Plant Cell Rep 34, 425–433 (2015). https://doi.org/10.1007/s00299-014-1720-6

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  • DOI: https://doi.org/10.1007/s00299-014-1720-6

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