Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50

BMC Biotechnology

Veronique Beiss³  Holger Spiegel³  Alexander Boes³  Matthias Scheuermayer¹  Andreas Reimann³  Stefan Schillberg³  Rainer Fischer² 

[1] Research Center for Infectious Diseases, University of Wuerzburg, Josef Schneider Str. 2/Bau D15, Wuerzburg, 97080, Germany

[2] RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, Aachen, 52074, Germany

[3] Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, Aachen, 52074, Germany

关键词: Plastid targeting;    Plant-made vaccines;    Agroinfiltration;    Gametes;    Sexual stage;    Plasmodium falciparum;   

Others  :  1234798
DOI  :  10.1186/s12896-015-0225-x

 received in 2015-08-13, accepted in 2015-11-24,  发布年份 2015

【 摘 要 】

Background

Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies.

Results

We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastid-targeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 μg/g recombinant protein per fresh leaf material for ER-retarded and16.2 μg/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG.

Conclusions

The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation.

【 授权许可】

   
2015 Beiss et al.

【 参考文献 】

• [1]Malaria Report. World Health Organization, Geneva, Switzerland; 2014.
• [2]Fairhurst RM, Nayyar GM, Breman JG, Hallett R, Vennerstrom JL, Duong S et al.. Artemisinin-resistant malaria: research challenges, opportunities, and public health implications. Am J Trop Med Hyg. 2012; 87(2):231-241.
• [3]Plowe CV. The evolution of drug-resistant malaria. Trans R Soc Trop Med Hyg. 2009; 103 Suppl 1:S11-14.
• [4]Targett GA, Greenwood BM. Malaria vaccines and their potential role in the elimination of malaria. Malaria J. 2008;7.
• [5]Murray CJL, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D et al.. Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet. 2012; 379(9814):413-431.
• [6]Mian-McCarthy S, Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Methogo BGNO et al.. A Phase 3 Trial of RTS, S/AS01 Malaria Vaccine in African Infants. New Engl J Med. 2012; 367(24):2284-2295.
• [7]Withers MR, McKinney D, Ogutu BR, Waitumbi JN, Milman JB, Apollo OJ et al. Safety and reactogenicity of an MSP-1 malaria vaccine candidate: A randomized phase Ib dose-escalation trial in Kenyan children. Plos Clin Trials. 2006;1(7).
• [8]Faber BW, Younis S, Remarque EJ, Garcia RR, Riasat V, Walraven V et al.. Diversity Covering AMA1-MSP1(19) Fusion Proteins as Malaria Vaccines. Infect Immun. 2013; 81(5):1479-1490.
• [9]Tamborrini M, Stoffel SA, Westerfeld N, Amacker M, Theisen M, Zurbriggen R et al. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations. Malaria J. 2011;10.
• [10]Wilby KJ, Lau TT, Gilchrist SE, Ensom MH. Mosquirix (RTS, S): A Novel Vaccine for the Prevention of Plasmodium falciparum Malaria. Ann Pharmacother. 2012; 46(3):384-393.
• [11]Tinto H, D'Alessandro U, Sorgho H, Valea I, Tahita MC, Kabore W et al.. Efficacy and safety of RTS, S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet. 2015; 386(9988):31-45.
• [12]Moorthy VS, Newman RD, Okwo-Bele JM. Malaria vaccine technology roadmap. Lancet. 2013; 382(9906):1700-1701.
• [13]Fries HCW, Lamers MBAC, Vandeursen J, Ponnudurai T, Meuwissen JHET. Biosynthesis of the 25-Kda Protein in the Macrogametes Zygotes of Plasmodium-Falciparum. Exp Parasitol. 1990; 71(2):229-235.
• [14]Kaslow DC, Quakyi IA, Syin C, Raum MG, Keister DB, Coligan JE et al.. A Vaccine Candidate from the Sexual Stage of Human Malaria That Contains Egf-Like Domains. Nature. 1988; 333(6168):74-76.
• [15]Kaslow DC, Bathurst IC, Isaacs SN, Keister DB, Moss B, Barr PJ. Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant Pfs25. Mem Inst Oswaldo Cruz. 1992; 87 Suppl 3:175-177.
• [16]Lobo CA, Dhar R, Kumar N. Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies. Infect Immun. 1999; 67(4):1688-1693.
• [17]Miura K, Keister DB, Muratova OV, Sattabongkot J, Long CA, Saul A. Transmission-blocking activity induced by malaria vaccine candidates Pfs25/Pvs25 is a direct and predictable function of antibody titer. Malaria J. 2007;6.
• [18]Wu YM, Ellis RD, Shaffer D, Fontes E, Malkin EM, Mahanty S et al. Phase 1 Trial of Malaria Transmission Blocking Vaccine Candidates Pfs25 and Pvs25 Formulated with Montanide ISA 51. Plos One. 2008;3(7).
• [19]Bousema T, Roeffen W, Meijerink H, Mwerinde H, Mwakalinga S, van Gemert GJ et al.. The dynamics of naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs230 & Pfs48/45 in a low endemic area in Tanzania. Plos One. 2010; 5(11):e14114.
• [20]Baum J, Richard D, Healer J, Rug M, Krnajski Z, Gilberger TW et al.. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. J Biol Chem. 2006; 281(8):5197-5208.
• [21]Yeoman JA, Hanssen E, Maier AG, Klonis N, Maco B, Baum J et al.. Tracking Glideosome-Associated Protein 50 Reveals the Development and Organization of the Inner Membrane Complex of Plasmodium falciparum. Eukaryot Cell. 2011; 10(4):556-564.
• [22]Frenal K, Polonais V, Marq JB, Stratmann R, Limenitakis J, Soldati-Favre D. Functional Dissection of the Apicomplexan Glideosome Molecular Architecture. Cell Host Microbe. 2010; 8(4):343-357.
• [23]Sanders PR, Cantin GT, Greenbaum DC, Gilson PR, Nebl T, Moritz RL et al.. Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts. Mol Biochem Parasit. 2007; 154(2):148-157.
• [24]Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R et al.. Malaria Parasites Co-opt Human Factor H to Prevent Complement-Mediated Lysis in the Mosquito Midgut. Cell Host Microbe. 2013; 13(1):29-41.
• [25]Fischer R, Schillberg S, Buyel JF, Twyman RM. Commercial Aspects of Pharmaceutical Protein Production in Plants. Curr Pharm Design. 2013; 19(31):5471-5477.
• [26]Phoolcharoen W, Bhoo SH, Lai HF, Ma JL, Arntzen CJ, Chen Q et al.. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J. 2011; 9(7):807-816.
• [27]Geyer BC, Muralidharan M, Cherni I, Doran J, Fletcher SP, Evron T et al.. Purification of transgenic plantderived recombinant human acetylcholinesterase-R. Chem Biol Interact. 2005; 157–158:331-334.
• [28]Boes A, Spiegel H, Edgue G, Kapelski S, Scheuermayer M, Fendel R et al.. Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits. Plant Biotechnol J. 2015; 13(2):222-234.
• [29]Farrance CE, Chichester JA, Musiychuk K, Shamloul M, Rhee A, Manceva SD et al.. Antibodies to plantproduced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity. Hum Vaccines. 2011; 7:191-198.
• [30]Farrance CE, Rhee A, Jones RM, Musiychuk K, Shamloul M, Sharma S et al.. A Plant-Produced Pfs230 Vaccine Candidate Blocks Transmission of Plasmodium falciparum. Clin Vaccine Immunol. 2011; 18(8):1351-1357.
• [31]Feller T, Thom P, Koch N, Spiegel H, Addai-Mensah O, Fischer R et al. Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate. Plos One. 2013;8(11).
• [32]Ma C, Wang LN, Webster DE, Campbell AE, Coppel RL. Production, characterisation and immunogenicity of a plant-made Plasmodium antigen-the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1. Appl Microbiol Biot. 2012; 94(1):151-161.
• [33]Voepel N, Boes A, Edgue G, Beiss V, Kapelski S, Reimann A et al.. Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants. Biotechnol J. 2014; 9(11):1435-1445.
• [34]Twyman RM, Schillberg S, Fischer R. Optimizing the Yield of Recombinant Pharmaceutical Proteins in Plants. Curr Pharm Design. 2013; 19(31):5486-5494.
• [35]Hoppmann V, Di Fiore S, Zimmermann S, Emans N, Rademacher T, Fischer R et al.. The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids. J Plant Physiol. 2002; 159(10):1061-1067.
• [36]Beiss V, Spiegel H, Boes A, Kapelski S, Scheuermayer M, Edgue G et al.. Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein. Biotechnol Bioeng. 2015; 112(7):1297-1305.
• [37]Bosch J, Paige MH, Vaidya AB, Bergman LW, Hol WGJ. Crystal structure of GAP50, the anchor of the invasion machinery in the inner membrane complex of Plasmodium falciparum. J Struct Biol. 2012; 178(1):61-73.
• [38]Di Fiore S, Li QR, Leech MJ, Schuster F, Emans N, Fischer R et al.. Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype. Plant Physiol. 2002; 129(3):1160-1169.
• [39]Di Fiore S, Hoppmann V, Fischer R, Schillberg S. Transient gene expression of recombinant terpenoid indole alkaloid enzymes in Catharanthus roseus leaves. Plant Mol Biol Rep. 2004; 22(1):15-22.
• [40]Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci. 2009; 14(12):669-679.
• [41]Meyers A, Chakauya E, Shephard E, Tanzer FL, Maclean J, Lynch A et al. Expression of HIV-1 antigens in plants as potential subunit vaccines. Bmc Biotechnol. 2008;8.
• [42]Buyel JF, Fischer R. Processing heterogeneous biomass Overcoming the hurdles in model building. Bioengineered. 2013; 4(1):21-24.
• [43]Buyel JF, Gruchow HM, Boes A, Fischer R. Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco. Biochem Eng J. 2014; 88:162-170.
• [44]Tuse D, Tu T, McDonald KA. Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes. Biomed Res Int. 2014; 2014:256135.
• [45]Wilken LR, Nikolov ZL. Downstream Processing of Transgenic Plant Systems: Protein Recovery and Purification Strategies. Molecular Farming in Plants: Recent Advances and Future Prospects 2012:217–257.
• [46]Tediosi F, Hutton G, Maire N, Smith TA, Ross A, Tanner M. Predicting the cost-effectiveness of introducing a pre-erythrocytic malaria vaccine into the expanded program on immunization in Tanzania. American Journal of Tropical Medicine and Hygiene. 2006; 75(2):131-143.
• [47]Seo MK, Baker P, Ngo KNL. Cost-effectiveness analysis of vaccinating children in Malawi with RTS,S vaccines in comparison with long-lasting insecticide-treated nets. Malaria J. 2014, 13.
• [48]Tediosi F, Maire N, Penny M, Studer A, Smith TA. Simulation of the cost-effectiveness of malaria vaccines. Malar J. 2009; 8:127. BioMed Central Full Text
• [49]von Itzstein M, Plebanski M, Cooke BM, Coppel RL. Hot, sweet and sticky: the glycobiology of Plasmodium falciparum. Trends Parasitol. 2008; 24(5):210-218.
• [50]Barr PJ, Green KM, Gibson HL, Bathurst IC, Quakyi IA, Kaslow DC. Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals. J Exp Med. 1991; 174(5):1203-1208.
• [51]Remarque EJ, Faber BW, Kocken CHM, Thomas AW. A diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbits. Infect Immun. 2008; 76(6):2660-2670.
• [52]Gregory JA, Li F, Tomosada LM, Cox CJ, Topol AB, Vinetz JM et al.. Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. Plos One. 2012; 7(5):e37179.
• [53]Dauvillee D, Delhaye S, Gruyer S, Slomianny C, Moretz SE, d'Hulst C et al. Engineering the Chloroplast Targeted Malarial Vaccine Antigens in Chlamydomonas Starch Granules. Plos One. 2010;5(12).
• [54]Scotti N, Rybicki EP. Virus-like particles produced in plants as potential vaccines. Expert Rev Vaccines. 2013; 12(2):211-224.
• [55]Koncz C, Schell J. The Promoter of Tl-DNA Gene 5 Controls the Tissue-Specific Expression of Chimeric Genes Carried by a Novel Type of Agrobacterium Binary Vector. Mol Gen Genet. 1986; 204(3):383-396.
• [56]Wirth CC, Glushakova S, Scheuermayer M, Repnik U, Garg S, Schaack D et al.. Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes. Cell Microbiol. 2014; 16(5):709-733.
• [57]Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F et al.. Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc Nglycans. Plant Biotechnol J. 2008; 6(2):189-201.
• [58]Wandelt CI, Khan MRI, Craig S, Schroeder HE, Spencer D, Higgins TJV. Vicilin with Carboxy-Terminal Kdel Is Retained in the Endoplasmic-Reticulum and Accumulates to High-Levels in the Leaves of Transgenic Plants. Plant J. 1992; 2(2):181-192.
• [59]Nolke G, Houdelet M, Kreuzaler F, Peterhansel C, Schillberg S. The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield. Plant Biotechnol J. 2014; 12(6):734-742.
• [60]Voss A, Niersbach M, Hain R, Hirsch HJ, Liao YC, Kreuzaler F et al.. Reduced Virus Infectivity in Nicotiana-Tabacum Secreting a Tmv-Specific Full-Size Antibody. Mol Breeding. 1995; 1(1):39-50.