Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures

Abstract

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett–Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Keywords

Daucus carota - Apiaceae - optimized medium culture - oral carrot-based vaccine - response surface methodology - synucleinopathies

Publication History

Received: 22 December 2023

Accepted after revision: 10 April 2024

Article published online:
02 May 2024

• References

• 1 Mendoza-Velásquez JJ, Flores-Vázquez JF, Barrón-Velázquez E, Sosa-Ortiz AL, Illigens BW, Siepmann T. Autonomic dysfunction in α-synucleinopathies. Front Neurol 2019; 10: 363
• 2 Savica R, Grossardt BR, Bower JH, Ahlskog JE, Rocca WA. Incidence and pathology of synucleinopathies and tauopathies related to parkinsonism. JAMA Neurol 2013; 70: 859-866
• 3 Dorsey ER, Bloem BR. The Parkinson pandemic-A call to action. JAMA Neurol 2018; 75: 9-10
• 4 Villar-Piqué A, Lopes da Fonseca T, Outeiro TF. Structure, function and toxicity of alpha-synuclein: The Bermuda triangle in synucleinopathies. J Neurochem 2016; 139 (Suppl. 1) 240-255
• 5 Wang Z, Gao G, Duan C, Yang H. Progress of immunotherapy of anti-α-synuclein in Parkinsonʼs disease. Biomed Pharmacother 2019; 115: 108843
• 6 Jankovic J, Tan EK. Parkinsonʼs disease: Etiopathogenesis and treatment. J Neurol Neurosurg Psychiatry 2020; 91: 795-808
• 7 Ghochikyan A, Petrushina I, Davtyan H, Hovakimyan A, Saing T, Davtyan A, Cribbs DH, Agadjanyan MG. Immunogenicity of epitope vaccines targeting different B cell antigenic determinants of human α-synuclein: Feasibility study. Neurosci Lett 2014; 560: 86-91
• 8 Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv 2011; 29: 210-222
• 9 Kurup VM, Thomas J. Edible vaccines: Promises and challenges. Mol Biotechnol 2020; 62: 79-90
• 10 Chen WP, Punja ZK. Transgenic herbicide and disease tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation. Plant Cell Rep 2002; 20: 929-935
• 11 Jayaraj J, Devlin R, Punja Z. Metabolic engineering of novel ketocarotenoid production in carrot plants. Transgenic Res 2008; 17: 489-501
• 12 Takaichi M, Oeda K. Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci. Plant Sci 2000; 153: 135-144
• 13 Rosales-Mendoza S, Tello-Olea MA. Carrot cells: A pioneering platform for biopharmaceuticals production. Mol Biotechnol 2015; 57: 219-232
• 14 Tekoah Y, Shulman A, Kizhner T, Ruderfer I, Fux L, Nataf Y, Bartfeld D, Ariel T, Gingis-Velitski S, Hanania U, Shaaltiel Y. Large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience. Plant Biotechnol J 2015; 13: 1199-1208
• 15 Ullisch DA, Muller CA, Maibaum S, Kirchhoff J, Schiermeyer A, Schillberg S, Roberts JL, Treffenfeldt W, Buchs J. Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization. J Biosci Bioeng 2012; 113: 242-248
• 16 Vaidya BK, Mutalik SR, Joshi RM, Nene SN, Kulkarni BD. Enhanced production of amidase from Rhodococcus erythropolis MTCC 1526 by medium optimisation using a statistical experimental design. J Ind Microbiol Biotechnol 2009; 36: 671-678
• 17 Montgomery DC. Design and Analysis of Experiments: Response Surface Method and Designs. New Jersey: John Wiley and Sons, Inc; 2005
• 18 Gorret N, bin Rosli SK, Oppenheim SF, Willis LB, Lessard PA, Rha CK, Sinskey AJ. Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production. J Biotechnol 2004; 108: 253-263
• 19 Holland T, Sack M, Rademacher T, Schmale K, Altmann F, Stadlmann J, Fischer R, Hellwig S. Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture. Biotechnol Bioeng 2010; 107: 278-289
• 20 Pinton R, Tomasi N, Zanin L. Molecular and physiological interactions of urea and nitrate uptake in plants. Plant Signal Behav 2016; 11: e1076603
• 21 Gerendás J, Zhu Z, Sattelmacher B. Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.). J Exp Bot 1998; 49: 1545-1554
• 22 Mérigout P, Lelandais M, Bitton F, Renou JP, Briand X, Meyer C, Daniel-Vedele F. Physiological and transcriptomic aspects of urea uptake and assimilation in Arabidopsis plants. Plant Physiol 2008; 147: 1225-1238
• 23 Garnica M, Houdusse F, Zamarreño AM, Garcia-Mina JM. Nitrate modifies the assimilation pattern of ammonium and urea in wheat seedlings. J Sci Food Agric 2010; 90: 357-369
• 24 Gamborg OL. The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiol 1970; 45: 372-375
• 25 Behrend J, Mateles RI. Nitrogen metabolism in plant cell suspension cultures I. Effect of amino acids on growth. Plant Physiol 1975; 56: 584-589
• 26 Claparols I, Santos MA, Torné JM. Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content. Plant Cell Tiss Organ Cult 1993; 34: 1-11
• 27 Carreño-Campos C, Arevalo-Villalobos JI, Villarreal ML, Ortiz-Caltempa A, Rosales-Mendoza S. Establishment of the carrot-made LTB-Syn antigen cell line in shake flask and airlift bioreactor cultures. Planta Med 2022; 88: 1060-1068
• 28 Vasilev N, Grömping U, Lipperts A, Raven N, Fischer R, Schillberg S. Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J 2013; 11: 867-874
• 29 Rasche S, Herwartz D, Schuster F, Jablonka N, Weber A, Fischer R, Schillberg S. More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments. Sci Rep 2016; 6: 23371
• 30 Martínez ME, Jorquera L, Poirrier P, Díaz K, Chamy R. Effect of Inoculum size and age, and sucrose concentration on cell growth to promote metabolites production in cultured Taraxacum officinale (Weber) cells. Plants (Basel) 2023; 12: 1116
• 31 Pavlov A, Georgiev MI, Ilieva M. Production of rosmarinic acid by Lavandula vera cell suspension in bioreactor: Effect of dissolved oxygen concentration and agitation. World J Microbiol Biotechnol 2005; 21: 389-392
• 32 Shohael AM, Murthy HN, Paek KY. Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides. Biotechnol Lett 2014; 36: 1727-1733
• 33 Park YJ, Han JE, Lee H, Jung YJ, Murthy HN, Park SY. Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors. AMB Express 2020; 10: 140
• 34 Thanh NT, Murthy HN, Yu KW, Seung Jeong C, Hahn EJ, Paek KY. Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng. J Plant Physiol 2006; 163: 1337-1341
• 35 Thanh NT, Murthy HN, Paek KY. Optimization of ginseng cell culture in airlift bioreactors and developing the large-scale production system. Ind Crops Prod 2014; 60: 343-348
• 36 Chun JH, Adhikari PB, Park SB, Han JY, Choi YE. Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47. Plant Cell Rep 2015; 34: 1551-1560
• 37 Navia-Osorio A, Garden H, Cusidó RM, Palazón J, Alfermann AW, Piñol MT. Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana. Planta Med 2002; 68: 336-340
• 38 Hayden CA, Egelkrout EM, Moscoso AM, Enrique C, Keener TK, Jimenez-Flores R, Wong JC, Howard JA. Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnol J 2012; 10: 979-984
• 39 Pniewski T, Kapusta J, Bociąg P, Kostrzak A, Fedorowicz-Strońska O, Czyż M, Gdula M, Krajewski P, Wolko B, Płucienniczak A. Plant expression, lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation. Plant Cell Rep 2012; 31: 585-595
• 40 Bolaños-Martínez OC, Govea-Alonso DO, Fragoso G, Sciutto E, Rosales-Mendoza S. Carrot cells expressing the VP1 and VP2 poliovirus proteins effectively elicited mucosal immunity. Plant Cell Tiss Organ Cult 2022; 148: 545-556
• 41 Uvarova EA, Belavin PA, Permyakova NV, Zagorskaya AA, Nosareva O, Kakimzhanova AA, Deineko EV. Oral immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10. Biomed Res Int 2013; 2013: 316304
• 42 Govea-Alonso DO, Tello-Olea MA, Beltrán-López J, Monreal-Escalante E, Salazar-González JA, Bañuelos-Hernández B, Rosales-Mendoza S. Assessment of carrot callus as biofactories of an atherosclerosis oral vaccine prototype. Mol Biotechnol 2017; 59: 482-489
• 43 Lionnet A, Leclair-Visonneau L, Neunlist M, Murayama S, Takao M, Adler CH, Derkinderen P, Beach TG. Does Parkinsonʼs disease start in the gut?. Acta Neuropathol 2018; 135: 1-12
• 44 Yang Q, Liang Q, Balakrishnan B, Belobrajdic DP, Feng QJ, Zhang W. Role of dietary nutrients in the modulation of gut microbiota: A narrative review. Nutrients 2020; 12 (02) 381
• 45 Barichella M, Pacchetti C, Bolliri C, Cassani E, Iorio L, Pusani C, Pinelli G, Privitera G, Cesari I, Faierman SA, Caccialanza R, Pezzoli G, Cereda E. Probiotics and prebiotic fiber for constipation associated with Parkinson disease: An RCT. Neurology 2016; 87: 1274-1280
• 46 Lubomski M, Xu X, Holmes AJ, Yang JYH, Sue CM, Davis RL. The impact of device-assisted therapies on the gut microbiome in Parkinsonʼs disease. J Neurol 2022; 269: 780-795
• 47 Wilken LR, Nikolov ZL. Recovery and purification of plant-made recombinant proteins. Biotechnol Adv 2012; 30: 419-433
• 48 Arevalo-Villalobos JI, Govea Alonso DO, Rosales-Mendoza S. Using carrot cells as biofactories and oral delivery vehicles of LTB-Syn: A low-cost vaccine candidate against synucleinopathies. J Biotechnol 2020; 309: 75-80
• 49 Trejo-Tapia G, Sepúlveda-Jiménez G, Trejo-Espino JL, Cerda-García-Rojas CM, de la Torre M, Rodríguez-Monroy M, Ramos-Valdivia AC. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst. Biotechnol Bioeng 2007; 98: 230-238
• 50 Dellaporta SL, Wood J, Hicks JB. A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1983; 1: 19-21
• 51 Arevalo-Villalobos JI, Govea-Alonso DO, Monreal-Escalante E, Zarazúa S, Rosales-Mendoza S. LTB-Syn: A recombinant immunogen for the development of plant-made vaccines against synucleinopathies. Planta 2017; 245: 1231-1239
• 52 Morales-Aguilar M, Bolaños-Martínez OC, Maldonado AR, Govea-Alonso DO, Carreño-Campos C, Villarreal ML, Rosales-Mendoza S, Ortiz-Caltempa A. Establishment of the Daucus carota SMC-1 cell suspension line for poliovirus vaccine development. Planta Med [Epub 2023 October 18]