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In vitro production of infectious Plasmodium falciparum sporozoites

Nature volume 612, pages 534–539 (2022)Cite this article

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Abstract

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1,2,3,4,5,6,7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

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Fig. 1: Late liver stage parasites 6 days after adding 5 × 104 iPfSPZ or mPfSPZ to triplicate wells of HC-04 cells or PHHs.
Fig. 2: Infection by intravenous injection of a human-liver chimeric FRGhuHep mouse with iPfSPZ or mPfSPZ.
Fig. 3: Gene expression in iPfSPZ versus mPfSPZ.

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Data availability

The NCBI BioProject accession number for the RNA-seq fastq files generated in this study is PRJNA753927. Details on how to use the files are provided in Supplementary Table 4. Genomes were obtained from PlasmoDB v.32 (https://plasmodb.org/plasmo/app/downloads/release-32/Pfalciparum3D7/); BDGP6.32, https://nov2020.archive.ensembl.org/Drosophila_melanogaster/Info/Index); and UC Irvine for A. stephensi (v.1.0, https://www.ncbi.nlm.nih.gov/genome/2653?genome_assembly_id=985930).

References

  1. Hoffman, S. L. et al. Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria. Hum. Vaccin. 6, 97–106 (2010).

    Article  CAS  Google Scholar 

  2. Seder, R. A. et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341, 1359–1365 (2013).

    Article  ADS  CAS  Google Scholar 

  3. Epstein, J. E. et al. Protection against Plasmodium falciparum malaria by PfSPZ vaccine. JCI Insight 2, e89154 (2017).

    Article  Google Scholar 

  4. Mordmuller, B. et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 542, 445–449 (2017).

    Article  ADS  Google Scholar 

  5. Jongo, S. A. et al. Increase of dose associated with decrease in protection against controlled human malaria infection by PfSPZ vaccine in Tanzanian adults. Clin. Infect. Dis. 71, 2849–2857 (2020).

    Article  CAS  Google Scholar 

  6. Mwakingwe-Omari, A. et al. Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity. Nature 595, 289–294 (2021).

    Article  ADS  CAS  Google Scholar 

  7. Sissoko, M. S. et al. Safety and efficacy of a three-dose regimen of Plasmodium falciparum sporozoite vaccine in adults during an intense malaria transmission season in Mali: a randomised, controlled phase 1 trial. Lancet Infect. Dis. 22, 377–389 (2022).

    Article  CAS  Google Scholar 

  8. Trager, W. & Jensen, J. B. Human malaria parasites in continuous culture. Science 193, 673–675 (1976).

    Article  ADS  CAS  Google Scholar 

  9. Haynes, J. D., Diggs, C. L., Hines, F. A. & Desjardins, R. E. Culture of human malaria parasites Plasmodium falciparum. Nature 263, 767–769 (1976).

    Article  ADS  CAS  Google Scholar 

  10. Ifediba, T. & Vanderberg, J. P. Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature 294, 364–366 (1981).

    Article  ADS  CAS  Google Scholar 

  11. Campbell, C. C., Collins, W. E., Nguyen Dinh, P., Barber, A. & Broderson, J. R. Plasmodium falciparum gametocytes from culture in vitro develop to sporozoites that are infectious to primates. Science 217, 1048–1050 (1982).

    Article  ADS  CAS  Google Scholar 

  12. Warburg, A. & Schneider, I. In vitro culture of the mosquito stages of Plasmodium falciparum. Exp. Parasitol. 76, 121–126 (1993).

    Article  CAS  Google Scholar 

  13. World Malaria Report 2021 (WHO, 2021).

  14. Epstein, J. E. et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity. Science 334, 475–480 (2011).

    Article  ADS  CAS  Google Scholar 

  15. Ishizuka, A. S. et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat. Med. 22, 614–623 (2016).

    Article  CAS  Google Scholar 

  16. Sissoko, M. S. et al. Safety and efficacy of PfSPZ Vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, double-blind phase 1 trial. Lancet Infect. Dis. 17, 498–509 (2017).

    Article  CAS  Google Scholar 

  17. Lyke, K. E. et al. Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection. Proc. Natl Acad. Sci. USA 114, 2711–2716 (2017).

    Article  ADS  CAS  Google Scholar 

  18. Goswami, D. et al. A replication-competent late liver stage–attenuated human malaria parasite. JCI Insight 5, e135589 (2020).

    Article  Google Scholar 

  19. Warburg, A. & Miller, L. H. Sporogonic development of a malaria parasite in vitro. Science 255, 448–450 (1992).

    Article  ADS  CAS  Google Scholar 

  20. Al-Olayan, E. M., Beetsma, A. L., Butcher, G. A., Sinden, R. E. & Hurd, H. Complete development of mosquito phases of the malaria parasite in vitro. Science 295, 677–679 (2002).

    Article  ADS  CAS  Google Scholar 

  21. Porter-Kelley, J. M. et al. Plasmodium yoelii: axenic development of the parasite mosquito stages. Exp. Parasitol. 112, 99–108 (2006).

    Article  CAS  Google Scholar 

  22. Barr, P. J. et al. Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals. J. Exp. Med. 174, 1203–1208 (1991).

    Article  CAS  Google Scholar 

  23. Posthuma, G. et al. Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts. J. Cell Biol. 46, 18–24 (1988).

    CAS  Google Scholar 

  24. Benton, G., Arnaoutova, I., George, J., Kleinman, H. K. & Koblinski, J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Adv. Drug Deliv. Rev. 79-80, 3–18 (2014).

    Article  CAS  Google Scholar 

  25. Hoffman, S. L. et al. Sporozoite vaccine induces genetically restricted T cell elimination of malaria from hepatocytes. Science 244, 1078–1081 (1989).

    Article  ADS  CAS  Google Scholar 

  26. Doolan, D. L. & Hoffman, S. L. The complexity of protective immunity against liver-stage malaria. J. Immunol. 165, 1453–1462 (2000).

    Article  CAS  Google Scholar 

  27. Hoffman, S. L. & Doolan, D. L. Malaria vaccines-targeting infected hepatocytes. Nat. Med. 6, 1218–1219 (2000).

    Article  CAS  Google Scholar 

  28. Weiss, W. R. & Jiang, C. G. Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites. PLoS ONE 7, e31247 (2012).

    Article  ADS  CAS  Google Scholar 

  29. Camponovo, F. et al. Proteome-wide analysis of a malaria vaccine study reveals personalized humoral immune profiles in Tanzanian adults. eLife 9, e53080 (2020).

    Article  CAS  Google Scholar 

  30. Aly, A. S., Vaughan, A. M. & Kappe, S. H. Malaria parasite development in the mosquito and infection of the mammalian host. Annu. Rev. Microbiol. 63, 195–221 (2009).

    Article  CAS  Google Scholar 

  31. Longley, R. J. et al. Comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates. Sci. Rep. 5, 11820 (2015).

    Article  ADS  Google Scholar 

  32. Atella, G. C., Silva-Neto, M. A., Golodne, D. M., Arefin, S. & Shahabuddin, M. Anopheles gambiae lipophorin: characterization and role in lipid transport to developing oocyte. Insect Biochem. Mol. Biol. 36, 375–386 (2006).

    Article  CAS  Google Scholar 

  33. Costa, G. et al. Non-competitive resource exploitation within mosquito shapes within-host malaria infectivity and virulence. Nat. Commun. 9, 3474 (2018).

    Article  ADS  CAS  Google Scholar 

  34. Gare, D. C., Piertney, S. B. & Billingsley, P. F. Anopheles gambiae collagen IV genes: cloning, phylogeny and midgut expression associated with blood feeding and Plasmodium infection. Int. J. Parasitol. 33, 681–690 (2003).

    Article  CAS  Google Scholar 

  35. Nacer, A., Walker, K. & Hurd, H. Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi. Parasit. Vectors 1, 33 (2008).

    Article  Google Scholar 

  36. Ponnudurai, T., Meuwissen, J. H., Leeuwenberg, A. D., Verhave, J. P. & Lensen, A. H. The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 76, 242–250 (1982).

    Article  CAS  Google Scholar 

  37. Li, T. et al. Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum. Malar. J. 14, 150 (2015).

    Article  Google Scholar 

  38. Feldmann, A. M. & Ponnudurai, T. Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum. Med. Vet. Entomol. 3, 41–52 (1989).

    Article  CAS  Google Scholar 

  39. Bounkeua, V., Li, F. & Vinetz, J. M. In vitro generation of Plasmodium falciparum ookinetes. Am. J. Trop. Med. Hyg. 83, 1187–1194 (2010).

    Article  Google Scholar 

  40. FiberCell Systems Hollow Fiber Cell Culture: An Overview (FibreCellSystems); https://www.fibercellsystems.com/instructional-video-fibercell-systems-hollow-fiber-cell-culture-an-overview/ (2012).

  41. Operation of a FiberCell Systems Duet Pump (FibreCellSystems); https://www.fibercellsystems.com/instructional-video-operation-of-a-fibercell-systems-duet-pump/ (2013).

  42. Zavala, F., Gwadz, R. W., Collins, F. H., Nussenzweig, R. S. & Nussenzweig, V. Monoclonal antibodies to circumsporozoite proteins identify the species of malaria parasites in infected mosquitoes. Nature 299, 737–738 (1982).

    Article  ADS  CAS  Google Scholar 

  43. Roestenberg, M. et al. Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites. Am. J. Trop. Med. Hyg. 88, 5–13 (2013).

    Article  CAS  Google Scholar 

  44. Sattabongkot, J. et al. Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 74, 708–715 (2006).

    Article  CAS  Google Scholar 

  45. Holder, A. A. The carboxy-terminus of merozoite surface protein 1: structure, specific antibodies and immunity to malaria. Parasitology 136, 1445–1456 (2009).

    Article  CAS  Google Scholar 

  46. Tsuji, M., Mattei, D., Nussenzweig, R. S., Eichinger, D. & Zavala, F. Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites. Parasitol. Res. 80, 16–21 (1994).

    Article  CAS  Google Scholar 

  47. Sanchez, G. I., Rogers, W. O., Mellouk, S. & Hoffman, S. L. Plasmodium falciparum: exported protein-1, a blood stage antigen, is expressed in liver stage parasites. Exp. Parasitol. 79, 59–62 (1994).

    Article  CAS  Google Scholar 

  48. Guerin-Marchand, C. et al. A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning. Nature 329, 164–167 (1987).

    Article  ADS  CAS  Google Scholar 

  49. Vaughan, A. M. et al. Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. J. Clin. Invest. 122, 3618–3628 (2012).

    Article  CAS  Google Scholar 

  50. Kefi, M. et al. New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes. Sci. Rep. 8, 1462 (2018).

    Article  ADS  Google Scholar 

  51. Mensah, V. A. et al. Safety, immunogenicity and efficacy of prime-boost vaccination with ChAd63 and MVA encoding ME-TRAP against Plasmodium falciparum infection in adults in Senegal. PLoS ONE 11, e0167951 (2016).

    Article  Google Scholar 

  52. Zanghì, G. et al. A specific PfEMP1 is expressed in P. falciparum sporozoites and plays a role in hepatocyte infection. Cell Rep. 22, 2951–2963 (2018).

    Article  Google Scholar 

  53. Gardner, M. J. et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511 (2002).

    Article  ADS  CAS  Google Scholar 

  54. Celniker, S. E. et al. Finishing a whole-genome shotgun: release 3 of the Drosophila melanogaster euchromatic genome sequence. Genome Biol. 3, research0079.1 (2002).

    Article  Google Scholar 

  55. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  56. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  57. Wagner, G. P., Kin, K. & Lynch, V. J. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 131, 281–285 (2012).

    Article  CAS  Google Scholar 

  58. Breitling, R., Armengaud, P., Amtmann, A. & Herzyk, P. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett. 573, 83–92 (2004).

    Article  CAS  Google Scholar 

  59. Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).

    Article  ADS  CAS  MATH  Google Scholar 

  60. Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008).

    Article  MATH  Google Scholar 

  61. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  CAS  Google Scholar 

  62. Diehl, K. H. et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21, 15–23 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Warburg and E. H. Hurd for advice regarding in vitro culture; T. L. Richie for reading the manuscript; A. Vaughan for advice regarding infection of FRG-knockout FRGhuHep mice; A. J. Radtke for confocal imaging of oocysts; E. James for cryopreservation of PfSPZ; R. Shepherd and M. Thambi for iPfSPZ production; A. Belmonte for assistance with T cell studies; and the members of Sanaria’s gametocyte and mosquito production teams and experimental animal study teams for their support. This work was supported by funds from the NIAID, NIH under SBIR grants 1R43AI085740-01, 2R44AI085740-03A1 and 2R44AI085740-06A1, and the CDMRP Program, USA Medical Research Acquisition Activity under contract W81XWH-16-2-0025. RNA-seq studies were funded by seed funds from Seattle Children’s Research Institute to S.H.I.K. The views expressed in this article reflect the results of research conducted by the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government.

Author information

Author notes

  1. Benjamin U. Hoffman

    Present address: Department of Medicine, UCSF, San Francisco, CA, USA

  2. Hashani Hettiarachchi

    Present address: Heritage College of Osteopathic Medicine, Ohio University, Dublin, OH, USA

Authors and Affiliations

  1. Sanaria, Rockville, MD, USA

    Abraham G. Eappen, Tao Li, Meghan Marquette, Sumana Chakravarty, Natasha KC, Hashani Hettiarachchi, Asha Patil, Yonas Abebe, Christiane Tran, Alemtaye A. Yossef, Ian McWilliams, Ayyappan Rathakrishnan, Ehud Inbar, Ahmed S. I. Aly, Patricia De La Vega, Tint Wai, MingLin Li, Peter F. Billingsley, B. Kim Lee Sim & Stephen L. Hoffman

  2. Protein Potential, Rockville, MD, USA

    Natasha KC, Tint Wai, MingLin Li & B. Kim Lee Sim

  3. Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, USA

    Gigliola Zanghi, Robert D. Morrison & Stefan H. I. Kappe

  4. Columbia University Irving Medical Center, New York, NY, USA

    Benjamin U. Hoffman

  5. Malaria Department, Naval Medical Research Center, Silver Spring, MD, USA

    Maria Belmonte & Martha Sedegah

  6. Henry M. Jackson Foundation, Bethesda, MD, USA

    Maria Belmonte

  7. Antigen Discovery Incorporated (ADI), Irvine, CA, USA

    Joseph J. Campo

  8. Institute for Bioscience and Biotechnology Research, Rockville, MD, USA

    Harley King

  9. Department of Pediatrics, University of Washington, Seattle, WA, USA

    Stefan H. I. Kappe

  10. Department of Global Health, University of Washington, Seattle, WA, USA

    Stefan H. I. Kappe

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  1. Abraham G. Eappen
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Contributions

S.L.H. led the entire project. A.G.E., S.C., P.F.B., B.K.L.S. and S.L.H. designed the studies. A.G.E., M.M., H.H., I.M. and A.A.Y. performed in vitro culture experiments and data collection. P.D.L.V., B.U.H. and A.R. performed in vitro infectivity assays using HC-04 cells and data collection. P.D.L.V., H.H., H.K. and A.G.E. performed in vitro infectivity assays using PHHs and data collection. S.C., N.K., T.L., C.T. and A.G.E. performed FRG mouse infection studies and data collection. T.L., A.P., Y.A., C.T. and B.K.L.S. produced stage V gametocytes. T.L., A.G.E., I.M., H.H., C.T., B.U.H. and S.C. performed P. falciparum life cycle without mosquitoes and data collection. G.Z., R.D.M. and S.H.I.K. performed transcriptome analysis and data collection. E.I. performed RT–qPCR. M.B. and M.S. performed T cell studies and data collection. S.C. and N.K. immunized mice. J.J.C. performed proteome assays. A.S.I.A., M.L. and T.W. performed immunoblot analysis. A.G.E., P.F.B., B.K.L.S. and S.L.H. wrote the manuscript. B.K.L.S. and S.L.H. supervised the project. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Stephen L. Hoffman.

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Competing interests

A.G.E., S.C., N.K, A.P., Y.A., A.A.Y., E.I., A.S.I.A., T.W., M.L., P.F.B., B.K.L.S. and S.L.H. are employees of Sanaria. B.K.L.S. and S.L.H. own stock in Sanaria. US patents on in vitro PfSPZ, 9878026B2 (2018) and 10441646B2 (2019) have been issued (inventors A.G.E. and S.L.H.). T.L., M.M., H.H., C.T., I.M., A.R. and P.D.V. were employees of Sanaria at the time the study was conducted.

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Extended data figures and tables

Extended Data Fig. 1 Ookinetes and oocysts in vitro.

a, b. In vitro transformed Pf zygotes and S2 feeder cells were added to Matrigel matrix coated wells of 8-well chamber slides and 24 h (a) or 36 (b) later an anti-Pfs25 mAb was added to the wells and the parasites were assessed by immunofluorescence assay. a. At 24 h, Pfs25 staining of an ookinete and the trail it had shed moving across the well are shown. b. At 36 h, Pfs25 staining of an early oocyst and the trail it had shed indicating the likely path the ookinete took before transforming to an oocyst are shown. Scale bars, 10 µm. Ookinetes expressing Pfs25 in 8-well chamber slide Matrigel cultures were identified in 3 different experiments. c. At 3 days (upper panels) an anti-Pfs25 mAb or at 8 days (lower panels) an anti-PfCSP mAb was added to the cultures and oocysts were assessed by immunofluorescence using confocal microscopy. Punctate localization in 8-day oocysts indicates budding PfSPZ. DAPI signals not localized to oocysts indicate feeder cell nuclei. In vitro produced 3-day oocysts expressing Pf25 and 8-day oocysts expressing PfCSP were detected in numerous experiments in 8-well chamber slides with Matrigel. d. A. stephensi mosquitoes were fed a blood meal containing stage V Pf gametocytes. At 3 days (upper panels) and 8 days (lower panels) the midguts were dissected and stained with anti-Pfs25 mAb (upper panels) and anti-PfCSP mAb (lower panels) and assessed by immunofluorescence using confocal microscopy. e. Comparison of conversion rates of gametocytes to iPfSPZ in different culture conditions. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. There were 7, 23, 24, 11, and 37 different independent experiments respectively for 8-well chamber slides with Sf21 cells, 8-well chamber slides with Matrigel, 12-well plates with S2 cells and collagen I, 12-well plates with S2 cells and GMP collagen, and 12-well plates with S2 cells and no matrix. All data were compared using Kruskal-Wallis test of median values (<0.0001), and post-hoc pairwise comparisons were made using Dunn’s multiple comparisons test. Significant differences and their respective p values are shown on the figure. f-g. In vitro transformed Pf zygotes (the product of 2x103 stage V gametocytes) with (f) and without (g) S2 feeder cells were added to Matrigel matrix coated wells of 8-well chamber slides. On day 15 post initiation of the culture, oocysts were assessed by IFA using an anti-PfCSP mAb. The culture including S2 cells (f) had many oocysts and iPfSPZ, indicated by arrows. The culture without S2 cells (g) had significantly lower numbers of oocysts (Supplementary Table 2). The oocysts in the cultures without S2 cells (g) had many budding PfSPZ expressing PfCSP (dots), but no fully developed iPfSPZ, suggesting an arrest in development. This experiment was performed three times and photomicrographs taken once.

Extended Data Fig. 2 Pf life cycle without mosquitoes or humans.

iPfSPZ were injected into mice containing humanized livers (FRGhuHep mice). Six and 7 days later the mice were transfused with human erythrocytes, and 6 h after the day 7 transfusion, blood was removed from the mice and cultured in vitro using standard methods. a, Parasites developed in these cultures to form Pf rings, trophozoites and schizonts; b. After induction, Pf gametocytes were produced in the cultures.

Extended Data Fig. 3 Gene and protein expression of iPfSPZ.

a. Scatter plot showing correlation of transcriptomes; x axis mPfSPZ, y axis iPfSPZ, right upper corner in red print, expressed liver stage genes. b. Heatmap of genes expressed in PfSPZ detected by RNASeq. 3,656 genes with ≥5 TPM in at least one sample are plotted. The raw gene expression reads from both iPfSPZ and mPfSPZ samples are in Supplementary Table 4. c. Comparative transcriptomics of mPfSPZ and iPfSPZ. Volcano plot of differentially expressed genes are plotted based upon average fold change and unadjusted average p-value from the 5 DE tools (significance of differential expression, see methods). Extreme low p-values are cropped at 1e-08 for plotting. Notable, differentially expressed genes are highlighted in red. d. PfCSP expression by mPfSPZ and iPfSPZ by immunoblot analysis. Lanes 1-3 are 2.0x105, 5.0x104 and 1.25x104 mPfSPZ, and lanes 5-7 are 1.1x106, 2.0x105, 5.0x104 iPfSPZ respectively. No sample was loaded in lane 4. Molecular size markers are indicated on the left.

Extended Data Fig. 4 T-cell responses in fresh and cryopreserved splenocytes.

Data show spot forming cells (SFC) expressing interferon gamma (IFNγ)/106 splenocytes from 6 mice immunized with iPfSPZ and 6 mice immunized with mPfSPZ at 2 weeks after the fourth dose of iPfSPZ or mPfSPZ. Splenocytes from 2 mice were pooled so that there were 3 samples from each group of immunized mice (a, b, c, d, e, f and h). Each pooled splenocyte sample was assayed in duplicate and each data point represents the mean of the duplicates. Results are median and interquartile ranges of SFC/106 splenocytes after incubation with stimulating cells at different concentrations. P values were calculated using the Mann-Whitney U test. The same symbol was used for each pool sample at the 3 different concentrations of stimulating cells. T-cell responses against Plasmodium falciparum-infected red blood cells (PfRBC) in fresh (a) and cryopreserved (b) splenocytes from immunized mice. One pool (●) had no detectable signal at 1x105 PfRBC but had a signal at both 2x105 PfRBC and 5x104 PfRBC. The negative response at 5x104 was considered a technical error and removed from the analysis. c, d. T-cell responses against mPfSPZ in fresh (c) and cryopreserved (d) splenocytes from immunized mice. Splenocytes were stimulated with 2.5x104 mPfSPZ. The responses to incubation with vaccine diluent alone are also shown. e, f. T-cell responses against uninfected red blood cell (uRBC) in fresh (E) and cryopreserved (F) splenocytes from immunized mice. The same symbol was used for each pool at the 3 different concentrations of uRBC (2x105, 1x105, and 5x104). g. T-cell responses against mPfSPZ in fresh splenocytes from naïve mice. Splenocytes of 2 naïve mice were pooled and frozen before assaying in duplicates and splenocytes of 2 naïve mice were pooled. and assayed fresh in duplicate. Splenocytes from naïve mice were stimulated with 2.5x104 mPfSPZ. h. T-cell responses against PfRBC in fresh and cryopreserved splenocytes from naïve mice. Fresh and cryopreserved splenocytes from naïve mice were stimulated with 2x105, 1x105, and 5x104 PfRBCs.

Extended Data Table 1 Transformation efficiency of stage V gametocytes to PfSPZ in mosquitoes and in vitro culture
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Extended Data Table 2 Transformation efficiency of gametocytes to 8-day oocysts in in vitro was 39-fold more as compared to in vivo growth and development in mosquitoes
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Extended Data Table 3 iPfSPZ produced in 12-well culture plates with S2 cells and no matrix
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Extended Data Table 4 Infectivity in HC-04 cells of Matrigel matrix-produced iPfSPZ compared to mPfSPZ
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Extended Data Table 5 Infectivity to HC-04 cells of rat tail collagen 1 matrix-produced iPfSPZ compared to mPfSPZ
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Extended Data Table 6 Pf life cycle from gametocytes to gametocytes without the use of mosquitoes or primates
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Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

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Supplementary Table 4

Raw gene expression reads from both iPfSPZ and mPfSPZ samples.

Supplementary Table 5

Relative abundance of the 50 most highly expressed mPfSPZ gene transcripts in iPfSPZ as determined by RNA-seq.

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Eappen, A.G., Li, T., Marquette, M. et al. In vitro production of infectious Plasmodium falciparum sporozoites. Nature 612, 534–539 (2022). https://doi.org/10.1038/s41586-022-05466-7

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