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Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites

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Abstract

Malaria is transmitted through the injection of Plasmodium sporozoites into the skin by Anopheles mosquitoes. The parasites first replicate within the liver before infecting red blood cells, which leads to the symptoms of the disease. Experimental immunization with attenuated sporozoites that arrest their development in the liver has been extensively investigated in rodent models and humans. Recent technological advances have included the capacity to cryopreserve sporozoites for injection, which has enabled a series of controlled studies on human infection with sporozoites. Here, we used a cryopreservation protocol to test the efficiency of genetically attenuated cryopreserved sporozoites for immunization of mice in comparison with freshly isolated controls. This showed that cryopreserved sporozoites are highly viable as judged by their capacity to migrate in vitro but show only 20% efficiency in liver infection, which impacts their capacity to generate protection of animals in immunization experiments.

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References

  • Alexander DL, Mital J, Ward GE, Bradley P, Boothroyd JC (2005) Identification of the moving junction complex of toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog 1:e17. https://doi.org/10.1371/journal.ppat.0010017

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, Mandomando I, Spiessens B, Guinovart C, Espasa M, Bassat Q, Aide P, Ofori-Anyinam O, Navia MM, Corachan S, Ceuppens M, Dubois MC, Demoitié MA, Dubovsky F, Menéndez C, Tornieporth N, Ripley Ballou W, Thompson R, Cohen J (2004) Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 364:1411–1420. https://doi.org/10.1016/S0140-6736(04)17223-1

    Article  PubMed  CAS  Google Scholar 

  • Amino R, Thiberge S, Martin B et al (2006) Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med 12:220–224. https://doi.org/10.1038/ni1134

    Article  PubMed  CAS  Google Scholar 

  • Bane KS, Lepper S, Kehrer J, Sattler JM, Singer M, Reinig M, Klug D, Heiss K, Baum J, Mueller AK, Frischknecht F (2016) The actin filament-binding protein coronin regulates motility in Plasmodium sporozoites. PLoS Pathog 12:e1005710. https://doi.org/10.1371/journal.ppat.1005710

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bijker EM, Borrmann S, Kappe SH, Mordmüller B, Sack BK, Khan SM (2015) Novel approaches to whole sporozoite vaccination against malaria. Vaccine 33:7462–7468. https://doi.org/10.1016/j.vaccine.2015.09.095

    Article  PubMed  CAS  Google Scholar 

  • Butler NS, Schmidt NW, Vaughan AM, Aly AS, Kappe SHI, Harty JT (2011) Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites. Cell Host Microbe 9:451–462. https://doi.org/10.1016/j.chom.2011.05.008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Carruthers VB, Sibley LD (1997) Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114–123

    PubMed  CAS  Google Scholar 

  • Carruthers VB, Sibley LD (1999) Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31:421–428

    Article  PubMed  CAS  Google Scholar 

  • Dubremetz JF (2007) Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell Microbiol 9:841–848. https://doi.org/10.1111/j.1462-5822.2007.00909.x

    Article  PubMed  CAS  Google Scholar 

  • Hegge S, Münter S, Steinbüchel M, Heiss K, Engel U, Matuschewski K, Frischknecht F (2010) Multistep adhesion of Plasmodium sporozoites. FASEB J 24:2222–2234. https://doi.org/10.1096/fj.09-148700

    Article  PubMed  CAS  Google Scholar 

  • Hegge S, Uhrig K, Streichfuss M, Kynast-Wolf G, Matuschewski K, Spatz JP, Frischknecht F (2012) Direct manipulation of malaria parasites with optical tweezers reveals distinct functions of Plasmodium surface proteins. ACS Nano 6:4648–4662. https://doi.org/10.1021/nn203616u

    Article  PubMed  CAS  Google Scholar 

  • Hellmann JK, Münter S, Kudryashev M, Schulz S, Heiss K, Müller AK, Matuschewski K, Spatz JP, Schwarz US, Frischknecht F (2011) Environmental constraints guide migration of malaria parasites during transmission. PLoS Pathog 7:e1002080. https://doi.org/10.1371/journal.ppat.1002080

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hellmann JK, Perschmann N, Spatz JP, Frischknecht F (2013) Tunable substrates unveil chemical complementation of a genetic cell migration defect. Adv Healthc Mater 2:1162–1169. https://doi.org/10.1002/adhm.201200426

    Article  PubMed  CAS  Google Scholar 

  • Hodgson SH, Ewer KJ, Bliss CM, Edwards NJ, Rampling T, Anagnostou NA, de Barra E, Havelock T, Bowyer G, Poulton ID, de Cassan S, Longley R, Illingworth JJ, Douglas AD, Mange PB, Collins KA, Roberts R, Gerry S, Berrie E, Moyle S, Colloca S, Cortese R, Sinden RE, Gilbert SC, Bejon P, Lawrie AM, Nicosia A, Faust SN, Hill AVS (2015) Evaluation of the efficacy of ChAd63-MVA vectored vaccines expressing circumsporozoite protein and ME-TRAP against controlled human malaria infection in malaria-naive individuals. J Infect Dis 211:1076–1086. https://doi.org/10.1093/infdis/jiu579

    Article  PubMed  CAS  Google Scholar 

  • Hoffman SL, Goh LML, Luke TC, Schneider I, le TP, Doolan DL, Sacci J, de la Vega P, Dowler M, Paul C, Gordon DM, Stoute JA, Church LWP, Sedegah M, Heppner DG, Ballou WR, Richie TL (2002) Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis 185:1155–1164. https://doi.org/10.1086/339409

    Article  PubMed  Google Scholar 

  • Hopp CS, Chiou K, Ragheb DRT, Salman AM, Khan SM, Liu AJ, Sinnis P (2015) Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition. elife 4. https://doi.org/10.7554/eLife.07789

  • Kennedy M, Fishbaugher ME, Vaughan AM, Patrapuvich R, Boonhok R, Yimamnuaychok N, Rezakhani N, Metzger P, Ponpuak M, Sattabongkot J, Kappe SH, Hume JCC, Lindner SE (2012) A rapid and scalable density gradient purification method for Plasmodium sporozoites. Malaria J 11:1–10. https://doi.org/10.1186/1475-2875-11-421

    Article  Google Scholar 

  • Kumar H, Sattler JM, Singer M, Heiss K, Reinig M, Hammerschmidt-Kamper C, Heussler V, Mueller AK, Frischknecht F (2016) Protective efficacy and safety of liver stage attenuated malaria parasites. Sci Rep 6:1–9. https://doi.org/10.1038/srep26824

    Article  CAS  Google Scholar 

  • Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, Kappe SHI (2007) Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection. Infect Immun 75:3758–3768. https://doi.org/10.1128/IAI.00225-07

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • malERA Consultative Group on Vaccines (2011) A research agenda for malaria eradication: vaccines. PLoS Med 8:e1000398. https://doi.org/10.1371/journal.pmed.1000398

    Article  Google Scholar 

  • Mordmüller B, Supan C, Sim KL, Gómez-Pérez GP, Ospina Salazar CL, Held J, Bolte S, Esen M, Tschan S, Joanny F, Lamsfus Calle C, Löhr SJZ, Lalremruata A, Gunasekera A, James ER, Billingsley PF, Richman A, Chakravarty S, Legarda A, Muñoz J, Antonijoan RM, Ballester MR, Hoffman SL, Alonso PL, Kremsner PG (2015) Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres. Malaria J 14:117. https://doi.org/10.1186/s12936-015-0628-0

    Article  Google Scholar 

  • Mordmüller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, Gmeiner M, Campo JJ, Esen M, Ruben AJ, Held J, Calle CL, Mengue JB, Gebru T, Ibáñez J, Sulyok M, James ER, Billingsley PF, Natasha KC, Manoj A, Murshedkar T, Gunasekera A, Eappen AG, Li T, Stafford RE, Li M, Felgner PL, Seder RA, Richie TL, Sim BKL, Hoffman SL, Kremsner PG (2017) Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 542:1–17. https://doi.org/10.1038/nature21060

    Article  CAS  Google Scholar 

  • Moreau CA, Bhargav SP, Kumar H, Quadt KA, Piirainen H, Strauss L, Kehrer J, Streichfuss M, Spatz JP, Wade RC, Kursula I, Frischknecht F (2017) A unique profilin-actin interface is important for malaria parasite motility. PLoS Pathog 13:e1006412. https://doi.org/10.1371/journal.ppat.1006412

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mueller A-K, Labaied M, Kappe SHI, Matuschewski K (2005) Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 433:164–167. https://doi.org/10.1038/nature03188

    Article  PubMed  CAS  Google Scholar 

  • Münter S, Sabass B, Selhuber-Unkel C, Kudryashev M, Hegge S, Engel U, Spatz JP, Matuschewski K, Schwarz US, Frischknecht F (2009) Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host Microbe 6:551–562. https://doi.org/10.1016/j.chom.2009.11.007

    Article  PubMed  CAS  Google Scholar 

  • Nussenzweig RS, Vanderberg J, Most H, Orton C (1967) Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 216:160–162

    Article  PubMed  CAS  Google Scholar 

  • Ono T, Cabrita-Santos L, Leitao R, Bettiol E, Purcell LA, Diaz-Pulido O, Andrews LB, Tadakuma T, Bhanot P, Mota MM, Rodriguez A (2008) Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection. PLoS Pathog 4:e1000008. https://doi.org/10.1371/journal.ppat.1000008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pelham RJ, Wang YL (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci U S A 94:13661–13665

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Prudêncio M, Rodriguez A, Mota MM (2006) The silent path to thousands of merozoites: the Plasmodium liver stage. Nature Rev Microbiol 4:849–856. https://doi.org/10.1038/nrmicro1529

    Article  CAS  Google Scholar 

  • Quadt KA, Streichfuss M, Moreau CA, Spatz JP, Frischknecht F (2016) Coupling of retrograde flow to force production during malaria parasite migration. ACS Nano 10:2091–2102. https://doi.org/10.1021/acsnano.5b06417

    Article  PubMed  CAS  Google Scholar 

  • Rampling T, Ewer KJ, Bowyer G, Bliss CM, Edwards NJ, Wright D, Payne RO, Venkatraman N, de Barra E, Snudden CM, Poulton ID, de Graaf H, Sukhtankar P, Roberts R, Ivinson K, Weltzin R, Rajkumar BY, Wille-Reece U, Lee CK, Ockenhouse CF, Sinden RE, Gerry S, Lawrie AM, Vekemans J, Morelle D, Lievens M, Ballou RW, Cooke GS, Faust SN, Gilbert S, Hill AVS (2016) Safety and high level efficacy of the combination malaria vaccine regimen of RTS,S/AS01B with chimpanzee adenovirus 63 and modified Vaccinia Ankara vectored vaccines expressing ME-TRAP. J Infect Dis 214:772–781. https://doi.org/10.1093/infdis/jiw244

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Roestenberg M, Bijker EM, Sim BKL et al (2013) Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites. Am J Trop Med Hyg 88:5–13. https://doi.org/10.4269/ajtmh.2012.12-0613

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019

    Article  PubMed  CAS  Google Scholar 

  • Seder RA, Chang L-J, Enama ME, Zephir KL, Sarwar UN, Gordon IJ, Holman LA, James ER, Billingsley PF, Gunasekera A, Richman A, Chakravarty S, Manoj A, Velmurugan S, Li M, Ruben AJ, Li T, Eappen AG, Stafford RE, Plummer SH, Hendel CS, Novik L, Costner PJM, Mendoza FH, Saunders JG, Nason MC, Richardson JH, Murphy J, Davidson SA, Richie TL, Sedegah M, Sutamihardja A, Fahle GA, Lyke KE, Laurens MB, Roederer M, Tewari K, Epstein JE, Sim BKL, Ledgerwood JE, Graham BS, Hoffman SL, the VRC 312 Study Team, DiGiovanni C, Williams P, Luongo N, Mitchell J, Florez MB, Larkin B, Berkowitz N, Wilson B, Clarke T, Vasilenko O, Yamshchikov G, Sitar S, Stanford L, Pittman I, Bailer RT, Casazza J, Decederfelt H, Starling J, Williams EC, Lau A, Antonara S, Brocious J, Kemp M, Inglese J, Dranchak P, Abot EN, Reyes S, Ganeshan H, Belmonte M, Huang J, Belmonte A, Komisar J, Abebe Y, Getachew Y, Patil A, Matheny S, Nelson K, Overby J, Pich V, Wen Y, Fan R, Fomumbod E, Awe A, Chakiath C, King M, Orozco MS, Murshedkar T, Padilla D, Jiang B, Gao L, KC N, Xu R, Adams M, Plowe C, Loblein H, Renehan PZ, Kunchai M, Diep L (2013) Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341:1359–1365. https://doi.org/10.1126/science.1241800

    Article  PubMed  CAS  Google Scholar 

  • Singer M, Frischknecht F (2017) Time for genome editing: next-generation attenuated malaria parasites. TRENDS Parasitol 33:202–213. https://doi.org/10.1016/j.pt.2016.09.012

    Article  PubMed  CAS  Google Scholar 

  • Singer M, Marshall J, Heiss K, Mair GR, Grimm D, Mueller AK, Frischknecht F (2015) Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal rare microhomology-mediated end joining. Genome Biol 16:249. https://doi.org/10.1186/s13059-015-0811-1

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Singh N, Barnes SJ, Jenwithisuk R, Sattabongkot J, Adams JH (2016) A simple and efficient method for cryopreservation and recovery of viable Plasmodium vivax and P. falciparum sporozoites. Parasitol Int 65:552–557. https://doi.org/10.1016/j.parint.2015.12.003

    Article  PubMed  CAS  Google Scholar 

  • Singh N, Barnes SJ, Kennedy S, Adams JH (2017) Experimental evaluation of cryopreservative solutions to maintain in vitro and in vivo infectivity of P. berghei sporozoites. PLoS One 12:e0177304. https://doi.org/10.1371/journal.pone.0177304

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sirima SB, Mordmüller B, Milligan P, Ngoa UA, Kironde F, Atuguba F, Tiono AB, Issifou S, Kaddumukasa M, Bangre O, Flach C, Christiansen M, Bang P, Chilengi R, Jepsen S, Kremsner PG, Theisen M, Ouédraogo A, Kargougou D, Nébié I, Débé S, Diarra A, Bougouma E, Hounkpatin AB, Adegnika AA, Lell B, Joanny F, Honkpehedji YJ, Agobe JCD, Esen M, Ajua A, Asoala V, Anyorigiya T, Ansah NA, Buwembo W, Mworozi E, Sekikubo M, Abubakar I, Bojang K, Noor R, Okech B, Ejigu DA (2016) A phase 2b randomized, controlled trial of the efficacy of the GMZ2 malaria vaccine in African children. Vaccine 34:4536–4542. https://doi.org/10.1016/j.vaccine.2016.07.041

    Article  PubMed  CAS  Google Scholar 

  • Spring M, Murphy J, Nielsen R, Dowler M, Bennett JW, Zarling S, Williams J, de la Vega P, Ware L, Komisar J, Polhemus M, Richie TL, Epstein J, Tamminga C, Chuang I, Richie N, O’Neil M, Heppner DG, Healer J, O’Neill M, Smithers H, Finney OC, Mikolajczak SA, Wang R, Cowman A, Ockenhouse C, Krzych U, Kappe SHI (2013) First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers. Vaccine 31:4975–4983. https://doi.org/10.1016/j.vaccine.2013.08.007

    Article  PubMed  Google Scholar 

  • Tavares J, Formaglio P, Thiberge S, Mordelet E, van Rooijen N, Medvinsky A, Ménard R, Amino R (2013) Role of host cell traversal by the malaria sporozoite during liver infection. J Exp Med 210:905–915. https://doi.org/10.1084/jem.20121130

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tse JR, Engler AJ (2010) Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol Chapter 10:Unit 10:16–10.16.16. https://doi.org/10.1002/0471143030.cb1016s47

    Article  Google Scholar 

  • van Schaijk BCL, Ploemen IHJ, Annoura T, Vos MW, Foquet L, van Gemert GJ, Chevalley-Maurel S, van de Vegte-Bolmer M, Sajid M, Franetich JF, Lorthiois A, Leroux-Roels G, Meuleman P, Hermsen CC, Mazier D, Hoffman SL, Janse CJ, Khan SM, Sauerwein RW (2014) A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites. elife 3. https://doi.org/10.7554/eLife.03582

  • Vaughan AM, Kappe SHI (2017a) Genetically attenuated malaria parasites as vaccines. Expert Rev Vaccines 16:765–767. https://doi.org/10.1080/14760584.2017.1341835

    Article  PubMed  CAS  Google Scholar 

  • Vaughan AM, Kappe SHI (2017b) Malaria parasite liver infection and exoerythrocytic biology. Cold Spring Harb Perspect Med 7. https://doi.org/10.1101/cshperspect.a025486

  • Whitelaw JA, Latorre-Barragan F, Gras S, Pall GS, Leung JM, Heaslip A, Egarter S, Andenmatten N, Nelson SR, Warshaw DM, Ward GE, Meissner M (2017) Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol 15(1):1. https://doi.org/10.1186/s12915-016-0343-5

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • World Health Organization (2017) World Malaria Report 2017:1–196

    Google Scholar 

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Acknowledgements

We thank Christian Sommerauer and Miriam Reinig for rearing mosquitoes and Mirko Singer for comments on the manuscript. The work was funded by grants from the German Research Foundation (SFB 1129, FR 2140/6-1), the Human Frontier Science Program (RGY0066), and the Volkswagen Foundation to FF.

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Correspondence to Friedrich Frischknecht.

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Prinz, H., Sattler, J.M., Roth, A. et al. Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites. Parasitol Res 117, 2487–2497 (2018). https://doi.org/10.1007/s00436-018-5937-0

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