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In vitro production of infectious Plasmodium falciparum sporozoites


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 Pfalciparum 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.

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 (; BDGP6.32,; and UC Irvine for A. stephensi (v.1.0,


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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

Authors and Affiliations



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
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
Extended Data Table 3 iPfSPZ produced in 12-well culture plates with S2 cells and no matrix
Extended Data Table 4 Infectivity in HC-04 cells of Matrigel matrix-produced iPfSPZ compared to mPfSPZ
Extended Data Table 5 Infectivity to HC-04 cells of rat tail collagen 1 matrix-produced iPfSPZ compared to mPfSPZ
Extended Data Table 6 Pf life cycle from gametocytes to gametocytes without the use of mosquitoes or primates

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Peer Review File

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).

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