Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

Journal name:
Nature
Volume:
542,
Pages:
445–449
Date published:
DOI:
doi:10.1038/nature21060
Received
Accepted
Published online

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites1. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes2, 3, 4; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’)5, 6; or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine7, 8, 9, 10 or mefloquine11 (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’12, 13) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac)14. Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge12, 13 at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

At a glance

Figures

  1. Protective efficacy of PfSPZ-CVac.
    Figure 1: Protective efficacy of PfSPZ-CVac.

    a, Proportion of controls and vaccinees who developed microscopically detectable parasitaemia after CHMI by DVI of 3.2 × 103 PfSPZ Challenge, 8–10 weeks after last immunization, and 7–9 weeks after last dose of chloroquine. Vaccinees received three doses of 3.2 × 103 (n = 9), 1.28 × 104 (n = 9), or 5.12 × 104 (n = 9) PfSPZ and controls (n = 13) received three doses of normal saline (vaccinees in yellow, placebo recipients in grey). b, Parasitaemia over time measured by qPCR. c, Proportion of controls and vaccinees who developed microscopically detectable parasitaemia after CHMI by DVI of 3.2 × 103 PfSPZ Challenge 70–72 days after last immunization, and 65–67 days after last dose of chloroquine. Vaccinees received three doses of 5.12 × 104 PfSPZ at 14-day (n = 9) and 5-day intervals (n = 9) PfSPZ and controls (n = 6) received three doses of normal saline (vaccinees in yellow, placebo recipients in grey). One vaccinee in the 5-day interval group and one control did not participate in the CHMI.

  2. Transient parasitaemia following vaccination.
    Figure 2: Transient parasitaemia following vaccination.

    a, Parasitaemia measured by qPCR in the three dosage groups after each immunization. The subjects who were protected and not protected against CHMI are in yellow and grey, respectively. b, Number of subjects positive per number injected, median peak parasite density, and day of peak parasite density after each dose of PfSPZ-CVac. *Technical problem with day 7 and 8 samples of one volunteer.

  3. Anti-plasmodial antibody responses in vaccinated volunteers.
    Figure 3: Anti-plasmodial antibody responses in vaccinated volunteers.

    Antibodies were assessed in sera taken before any immunizations (pre-immunization), two weeks following last immunization (post-immunization) and one day before CHMI (pre-CHMI). ad, Antibodies were assessed to PfCSP by ELISA (a); air-dried PfSPZ by automated immunofluorescence assay (b); live PfSPZ by inhibition of sporozoite invasion (c); and 7,455 Pf peptides on a proteome array (d). a, PfCSP ELISA results are reported as net optical density (OD) 1.0; reciprocal serum dilution at which the optical density was 1.0 in post-immunization or pre-CHMI sera minus the OD 1.0 in pre-immunization sera. All negative net values were assigned a value of 1. Values above the dashed line are considered positive. b, Automated immunofluorescence assay (aIFA) results are reported as arbitrary fluorescent units (AFU) 2 × 105; reciprocal serum dilution at which the AFU were 2 × 105 in post-immunization and pre-CHMI sera. c, Inhibition of sporozoite invasion values are reported as the reciprocal dilution of pre-immunization, post-immunization and pre-CHMI sera that inhibited by 75% the numbers of PfSPZ invading as compared to in negative controls without serum. d, The 22 proteins on the proteome array recognized by post-immunization sera from at least five volunteers from group III (highest dose) are delineated. The list is derived from bipartite graph analysis following normalization and background correction using values from sera taken before injection of PfSPZ Challenge in vaccinees and controls, and after injection of normal saline in controls. The threshold of positivity for the array studies was more conservative compared to the ELISA analyses; for example, for PfCSP, 5 out of 9 array-positive compared to 9 out of 9 ELISA-positive. In a–c, protected individuals are represented in yellow and unprotected ones in grey, and box plots display median (middle line), 25th (lower hinge) and 75th (upper hinge) quartile. Whiskers extend to values within 1.5× the inter-quartile ranges of the lower and upper hinges, respectively.

  4. T-cell immunogenicity and correlates of protection.
    Figure 4: T-cell immunogenicity and correlates of protection.

    a, b, Memory CD4 T cells producing IFN-γ, IL-2, and/or TNF-α following PfSPZ (a) or PfRBC stimulation (b). c, d, Memory CD8 T cells producing IFN-γ following PfSPZ (c) or PfRBC stimulation (d). For ad, results are the percentage of cytokine-producing cells after incubation with Pf antigen minus the percentage of cells after incubation with control antigen stimulation. e, Fold change compared to pre-immunization in the frequency of Vδ2+ T cells as a percentage of total lymphocytes. f, Cytokine polyfunctionality of PfSPZ- or PfRBC-specific memory CD4 T cells. Pie charts show the fraction of each cytokine combination out of the total cytokine response comparing subjects that were parasitaemic (+) or not parasitaemic (–) after CHMI. g, Individual data points for f, showing the composition of PfSPZ- (top) or PfRBC-specific (bottom) memory CD4 T cells producing any combination of IFN-γ, IL-2, and/or TNF-α at the time of CHMI. Subjects that remained without parasitaemia are shown in blue and subjects that developed parasitaemia are shown in grey. ae, The difference from pre-vaccine within a dose group was assessed by two-way ANOVA with Bonferroni correction. Open symbols denote parasitaemic after CHMI; closed symbols denote not parasitaemic after CHMI. f, Comparison between pie graphs was by a non-parametric partial permutation test; g, comparison between parasitaemic and not parasitaemic responses was by Wilcoxon test. Bars are median ± interquartile range (ad, g) or geometric mean and 95% confidence interval (e). Pre-imm, 3 days before first immunization; post-imm, 14 days after third immunization; pre-CHMI, 1 day before CHMI. *P < 0.05, **P < 0.01, ***P < 0.001.

  5. Distribution of adverse events.
    Extended Data Fig. 1: Distribution of adverse events.

    a, b, The number of adverse events (AEs) regardless of attribution to investigational product. Each bar represents one volunteer sorted on the number of adverse events from the time of first injection with normal saline (controls) or PfSPZ-CVac until the time of CHMI, approximately 17 weeks later (a) and adverse events in the same volunteers from initiation of CHMI until the end of follow-up (b). Mild (grade 1) adverse events are depicted in grey, moderate (grade 2) in yellow and severe (grade 3) in blue. Non-protected volunteers are marked with an ‘M’ on the x axis.

  6. CONSORT study flow chart.
    Extended Data Fig. 2: CONSORT study flow chart.
  7. CD4 T-cell cytokine polyfunctionality.
    Extended Data Fig. 3: CD4 T-cell cytokine polyfunctionality.

    PBMCs from subjects were drawn 14 days after third immunization (post-imm) or 1 day before CHMI (pre-CHMI), stimulated with PfSPZ, PfRBC, or stimulation controls, and stained for intracellular cytokine expression. a, b, The pie charts show the proportion of memory CD4 T cells expressing any combination of IFN-γ, IL-2, or TNF-α for each dose group after stimulation with PfSPZ (a) or PfRBC (b). Responses are background subtracted from control antigen stimulations 1% HSA or uninfected erythrocytes. c, d, The magnitude of the memory CD4 T-cell response for each combination of cytokines is shown in c and d. There is a trend towards higher polyfunctionality as dose increases. e, f, The median fluorescence intensity (MFI) for IFN-γ is shown for the different combination of IFN-γ+ cells following PfSPZ or PfRBC stimulation. Cells that simultaneously produce IFN-γ, IL-2, and TNF-α have the highest IFN-γ MFI.

  8. T-cell immunogenicity.
    Extended Data Fig. 4: T-cell immunogenicity.

    a, b, Memory CD4 T cells producing IL-4 (a) or IL-10 (b) after PfRBC stimulation. Memory γδ T cells producing IFN-γ, IL-2, or TNF-α following PfSPZ (c) or PfRBC stimulation (d). For ad, results are the percentage of cytokine-producing cells after incubation with PfSPZ minus the percentage of cells after incubation with vaccine diluent (medium with 1% HSA) as control or percentage of cytokine-producing cells after incubation with asexual Pf-infected red blood cells (PfRBC) minus uninfected RBCs as control. e, f, Total memory γδ T cells assessed before immunization (pre-imm) and 14 days after third immunization (post-imm) for the percentage of cells expressing CD38. The absolute frequencies are shown in e and the change from pre-vaccination to post-vaccination is shown in f. For ad, within a dose group, the difference from pre-vaccine was assessed by two-way ANOVA with Bonferroni correction. Data are median ± interquartile range. For e, f, difference from pre-vaccine was assessed by Wilcoxon signed rank test. P values were corrected for multiple comparisons by the Bonferroni method. *P < 0.05, *P < 0.01. Data are median ± interquartile range. Pre-imm, 3 days before first immunization; post-imm, 14 days after third immunization; pre-CHMI, 1 day before CHMI.

  9. Sub-family analysis of γδ T cells.
    Extended Data Fig. 5: Sub-family analysis of γδ T cells.

    af, The frequency of the circulating γδ T-cell subsets as a percentage of total lymphocytes was assessed in unstimulated PBMCs before the first immunization (pre-imm), 2 weeks after final immunization (post-imm), and the day before CHMI (pre-CHMI). Fold change compared to pre-imm is shown for total memory γδ T cells (a), Vγ9+Vδ2+ (b), Vγ9+Vδ1+ (c), Vγ9Vδ1+ (d), Vγ9+Vδ1Vδ2 (e), and Vγ9Vδ1Vδ2 (f) subfamilies. The frequency of Vγ9Vδ2+ subset is low to undetectable. Within a dose group, the difference from pre-imm was assessed by two-way ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01. Data are geometric mean ± 95% CI.

  10. Anti-plasmodial antibody responses in vaccinated volunteers who were immunized with three doses of 5.12 × 104 PfSPZ at 28-day, 14-day, or 5-day intervals.
    Extended Data Fig. 6: Anti-plasmodial antibody responses in vaccinated volunteers who were immunized with three doses of 5.12 × 104 PfSPZ at 28-day, 14-day, or 5-day intervals.

    Antibodies to PfCSP by ELISA were assessed in sera taken before any immunizations (pre-immunization), two weeks following last immunization (post-immunization) and 10 weeks after last immunization, which was one day before CHMI (pre-CHMI). PfCSP ELISA results are reported as net OD 1.0; the reciprocal serum dilution at which the optical density was 1.0 in post-immunization or pre-CHMI sera minus the OD 1.0 in pre-immunization sera. All values met criteria for positivity. Protected volunteers are represented by yellow circles and unprotected volunteers by grey circles.

  11. Development of PfSPZ Vaccine and PfSPZ-CVac in hepatocytes.
    Extended Data Fig. 7: Development of PfSPZ Vaccine and PfSPZ-CVac in hepatocytes.

    Radiation-attenuated PfSPZ in PfSPZ Vaccine invade hepatocytes and partially develop, but do not replicate. They are metabolically active and non-replicating. Infectious PfSPZ in PfSPZ-CVac invade hepatocytes and fully develop. A single PfSPZ replicates exponentially producing more than 104 merozoites. These merozoites are released into the circulation, and each merozoite can invade a different erythrocyte. Chloroquine prevents complete parasite development within erythrocytes, thereby preventing the development of merozoites that can invade new erythrocytes.

  12. Transient parasitaemia following vaccination at 5-day intervals.
    Extended Data Fig. 8: Transient parasitaemia following vaccination at 5-day intervals.

    Parasitaemia measured by qPCR over 22 days. The subjects who were protected and not protected against CHMI are in yellow and grey, respectively. The times of PfSPZ inoculations are shown as vertical red lines and the time of last CQ administration as a vertical blue line. CQ was given as 10 mg kg−1 (maximum 620 mg) loading dose on day 0 followed by 5 mg kg−1 (maximum 310 mg) chloroquine base on days 5, 10, and 15.

Tables

  1. Pf-specific T-cell correlates of sterile protection, stratified by group
    Extended Data Table 1: Pf-specific T-cell correlates of sterile protection, stratified by group

References

  1. malERA Consultative Group on Vaccines. A research agenda for malaria eradication: vaccines. PLoS Med. 8, e1000398 (2011)
  2. Clyde, D. F., Most, H., McCarthy, V. C. & Vanderberg, J. P. Immunization of man against sporozite-induced falciparum malaria. Am. J. Med. Sci. 266, 169177 (1973)
  3. Rieckmann, K. H., Carson, P. E., Beaudoin, R. L., Cassells, J. S. & Sell, K. W. Letter: Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg. 68, 258259 (1974)
  4. Hoffman, S. L. et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185, 11551164 (2002)
  5. Seder, R. A. et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341, 13591365 (2013)
  6. Ishizuka, A. S. et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat. Med. 22, 614623 (2016)
  7. Bijker, E. M. et al. Cytotoxic markers associate with protection against malaria in human volunteers immunized with Plasmodium falciparum sporozoites. J. Infect. Dis. 210, 16051615 (2014)
  8. Bijker, E. M. et al. Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc. Natl Acad. Sci. USA 110, 78627867 (2013)
  9. Roestenberg, M. et al. Protection against a malaria challenge by sporozoite inoculation. N. Engl. J. Med. 361, 468477 (2009)
  10. Roestenberg, M. et al. Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study. Lancet 377, 17701776 (2011)
  11. Bijker, E. M. et al. Sporozoite immunization of human volunteers under mefloquine prophylaxis is safe, immunogenic and protective: a double-blind randomized controlled clinical trial. PLoS One 9, e112910 (2014)
  12. Gómez-Pérez, G. P. et al. Controlled human malaria infection by intramuscular and direct venous inoculation of cryopreserved Plasmodium falciparum sporozoites in malaria-naive volunteers: effect of injection volume and dose on infectivity rates. Malar. J. 14, 306 (2015)
  13. Mordmüller, B. et al. Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres. Malar. J. 14, 117 (2015)
  14. Bastiaens, G. J. et al. Safety, immunogenicity, and protective efficacy of intradermal immunization with aseptic, purified, cryopreserved Plasmodium falciparum sporozoites in volunteers under chloroquine prophylaxis: a randomized controlled trial. Am. J. Trop. Med. Hyg. 94, 663673 (2016)
  15. Beaudoin, R. L., Strome, C. P. A., Mitchell, F. & Tubergen, T. A. Plasmodium berghei: immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen. Exp. Parasitol. 42, 15 (1977)
  16. Guerin-Marchand, C. et al. A liver-stage-specific antigen of Plasmodium falciparum characterized by gene cloning. Nature 329, 164167 (1987)
  17. Orito, Y. et al. Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation. Mol. Microbiol. 87, 6679 (2013)
  18. Tarun, A. S. et al. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc. Natl Acad. Sci. USA 105, 305310 (2008)
  19. Richie, T. L. et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines. Vaccine 33, 74527461 (2015)
  20. Epstein, J. E. et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity. Science 334, 475480 (2011)
  21. Suhrbier, A., Winger, L. A., Castellano, E. & Sinden, R. E. Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells. Infect. Immun. 58, 28342839 (1990)
  22. Behet, M. C. et al. Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum. Malar. J. 13, 136 (2014)
  23. Schofield, L. et al. γ Interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 330, 664666 (1987)
  24. Weiss, W. R., Sedegah, M., Beaudoin, R. L., Miller, L. H. & Good, M. F. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc. Natl Acad. Sci. USA 85, 573576 (1988)
  25. Doolan, D. L. & Hoffman, S. L. The complexity of protective immunity against liver-stage malaria. J. Immunol. 165, 14531462 (2000)
  26. Belnoue, E. et al. Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J. Immunol. 172, 24872495 (2004)
  27. Weiss, W. R. & Jiang, C. G. Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites. PLoS One 7, e31247 (2012)
  28. Fernandez-Ruiz, D. et al. Liver-resident memory CD8+ T cells form a front-line defense against malaria liver-stage infection. Immunity 45, 889902 (2016)
  29. Schats, R. et al. Heterologous protection against malaria after immunization with Plasmodium falciparum sporozoites. PLoS One 10, e0124243 (2015)
  30. Egan, J. E. et al. Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am. J. Trop. Med. Hyg. 49, 166173 (1993)
  31. Epstein, J. E. et al. Protection against Plasmodium falciparum malaria by PfSPZ Vaccine. JCI Insight 2, e89154 (2017)
  32. Lyke, K. et al. PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous malaria challenge. Proc. Natl Acad. Sci. USA (in the press)
  33. Sissoko, M. S. et al. Safety and efficacy of PfSPZ Vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed Malian adults: a randomised, double-blind trial. Lancet Infect Dis. (in the press)
  34. Roestenberg, M. et al. Controlled human malaria infections by intradermal injection of cryopreserved Plasmodium falciparum sporozoites. Am. J. Trop. Med. Hyg. 88, 513 (2013)
  35. Sahu, T. et al. Chloroquine neither eliminates liver stage parasites nor delays their development in a murine chemoprophylaxis vaccination model. Front. Microbiol. 6, 283 (2015)
  36. Fairley, N. H. Chemotherapeutic suppression and prophylaxis in malaria. Trans. R. Soc. Trop. Med. Hyg. 38, 311365 (1945)
  37. Planche, T. et al. Comparison of methods for the rapid laboratory assessment of children with malaria. Am. J. Trop. Med. Hyg. 65, 599602 (2001)
  38. Kamau, E., Alemayehu, S., Feghali, K. C., Saunders, D. & Ockenhouse, C. F. Multiplex qPCR for detection and absolute quantification of malaria. PLoS One 8, e71539 (2013)
  39. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611622 (2009)
  40. R Core Team. R: A Language and Environment for Statistical Computing. (2015)
  41. Roederer, M., Nozzi, J. L. & Nason, M. C. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 79, 167174 (2011)
  42. 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, 708715 (2006)
  43. Felgner, P. L. et al. Pre-erythrocytic antibody profiles induced by controlled human malaria infections in healthy volunteers under chloroquine prophylaxis. Sci. Rep. 3, 3549 (2013)
  44. Davies, D. H. et al. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery. Proc. Natl Acad. Sci. USA 102, 547552 (2005)
  45. Lamoreaux, L., Roederer, M. & Koup, R. Intracellular cytokine optimization and standard operating procedure. Nat. Protocols 1, 15071516 (2006)

Download references

Author information

  1. These authors contributed equally to this work.

    • Stephen L. Hoffman &
    • Peter G. Kremsner

Affiliations

  1. Institute of Tropical Medicine, University of Tübingen and German Center for Infection Research, partner site Tübingen, 72074 Tübingen, Germany

    • Benjamin Mordmüller,
    • Güzin Surat,
    • Heimo Lagler,
    • Albert Lalremruata,
    • Markus Gmeiner,
    • Meral Esen,
    • Jana Held,
    • Carlos Lamsfus Calle,
    • Juliana B. Mengue,
    • Tamirat Gebru,
    • Javier Ibáñez,
    • Mihály Sulyok &
    • Peter G. Kremsner
  2. Department of Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, 1090 Vienna, Austria

    • Heimo Lagler
  3. Sanaria Inc., Rockville, Maryland 20850, USA

    • Sumana Chakravarty,
    • Adam J. Ruben,
    • Eric R. James,
    • Peter F. Billingsley,
    • KC Natasha,
    • Anita Manoj,
    • Tooba Murshedkar,
    • Anusha Gunasekera,
    • Abraham G. Eappen,
    • Tao Li,
    • Richard E. Stafford,
    • Minglin Li,
    • Thomas L. Richie,
    • B. Kim Lee Sim &
    • Stephen L. Hoffman
  4. Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA

    • Andrew S. Ishizuka &
    • Robert A. Seder
  5. Antigen Discovery Inc., Irvine, California 92618, USA

    • Joseph J. Campo
  6. Protein Potential, LLC, Rockville, Maryland 20850, USA

    • KC Natasha,
    • Richard E. Stafford,
    • Minglin Li &
    • B. Kim Lee Sim
  7. Department of Medicine, University of California Irvine, Irvine, California 92697, USA

    • Phil L. Felgner

Contributions

B.M. designed the study, analysed the data, contributed to data collection and wrote the manuscript; B.K.L.S., E.R.J., A.J.R., A.G.E., T.L., R.E.S. and M.L manufactured the investigational products; A.M., R.S., T.M., A.G. and P.F.B. assured quality and regulatory compliance; C.L.C. and J.B.M. performed PfSPZ formulations; G.S., M.G., M.S. and H.L. collected data; A.L., M.E., J.I., T.G. and J.H. performed laboratory analyses; S.C., N.K, M.L. and A.J.R. performed and analysed all ELISA, IFA, and inhibition of sporozoite invasion studies; J.J.C. and P.L.F. performed and analysed protein array data; A.S.I. and R.A.S. performed and analysed cytometry experiments; T.L.R. oversaw the clinical trial; A.J.R., T.L.R., P.F.B., B.K.L.S. and S.L.H. analysed data; S.L.H. and P.G.K supervised the project, interpreted data, and wrote the manuscript. S.L.H. was the clinical trial sponsor representative and B.M. the principal investigator of the trial. S.L.H. and P.G.K. contributed equally to the work. All authors discussed the results and commented on the manuscript.

Competing financial interests

S.C., A.J.R., E.R.J., P.F.B., N.K, A.M., T.M., A.G., A.G.E., T.L., R.E.S, M.L., T.L.R., B.K.L.S. and S.L.H. are salaried employees of Sanaria Inc., the developer and owner of PfSPZ Challenge and the sponsor of the clinical trial. In addition, S.L.H. and B.K.L.S. have a financial interest in Sanaria Inc. J.J.C. is an Antigen Discovery Employee. P.L.F. owns stock and is a board member at Antigen Discovery, Inc. All other authors declare no conflicts of interest.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks L. Rénia and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Distribution of adverse events. (134 KB)

    a, b, The number of adverse events (AEs) regardless of attribution to investigational product. Each bar represents one volunteer sorted on the number of adverse events from the time of first injection with normal saline (controls) or PfSPZ-CVac until the time of CHMI, approximately 17 weeks later (a) and adverse events in the same volunteers from initiation of CHMI until the end of follow-up (b). Mild (grade 1) adverse events are depicted in grey, moderate (grade 2) in yellow and severe (grade 3) in blue. Non-protected volunteers are marked with an ‘M’ on the x axis.

  2. Extended Data Figure 2: CONSORT study flow chart. (155 KB)
  3. Extended Data Figure 3: CD4 T-cell cytokine polyfunctionality. (328 KB)

    PBMCs from subjects were drawn 14 days after third immunization (post-imm) or 1 day before CHMI (pre-CHMI), stimulated with PfSPZ, PfRBC, or stimulation controls, and stained for intracellular cytokine expression. a, b, The pie charts show the proportion of memory CD4 T cells expressing any combination of IFN-γ, IL-2, or TNF-α for each dose group after stimulation with PfSPZ (a) or PfRBC (b). Responses are background subtracted from control antigen stimulations 1% HSA or uninfected erythrocytes. c, d, The magnitude of the memory CD4 T-cell response for each combination of cytokines is shown in c and d. There is a trend towards higher polyfunctionality as dose increases. e, f, The median fluorescence intensity (MFI) for IFN-γ is shown for the different combination of IFN-γ+ cells following PfSPZ or PfRBC stimulation. Cells that simultaneously produce IFN-γ, IL-2, and TNF-α have the highest IFN-γ MFI.

  4. Extended Data Figure 4: T-cell immunogenicity. (229 KB)

    a, b, Memory CD4 T cells producing IL-4 (a) or IL-10 (b) after PfRBC stimulation. Memory γδ T cells producing IFN-γ, IL-2, or TNF-α following PfSPZ (c) or PfRBC stimulation (d). For ad, results are the percentage of cytokine-producing cells after incubation with PfSPZ minus the percentage of cells after incubation with vaccine diluent (medium with 1% HSA) as control or percentage of cytokine-producing cells after incubation with asexual Pf-infected red blood cells (PfRBC) minus uninfected RBCs as control. e, f, Total memory γδ T cells assessed before immunization (pre-imm) and 14 days after third immunization (post-imm) for the percentage of cells expressing CD38. The absolute frequencies are shown in e and the change from pre-vaccination to post-vaccination is shown in f. For ad, within a dose group, the difference from pre-vaccine was assessed by two-way ANOVA with Bonferroni correction. Data are median ± interquartile range. For e, f, difference from pre-vaccine was assessed by Wilcoxon signed rank test. P values were corrected for multiple comparisons by the Bonferroni method. *P < 0.05, *P < 0.01. Data are median ± interquartile range. Pre-imm, 3 days before first immunization; post-imm, 14 days after third immunization; pre-CHMI, 1 day before CHMI.

  5. Extended Data Figure 5: Sub-family analysis of γδ T cells. (258 KB)

    af, The frequency of the circulating γδ T-cell subsets as a percentage of total lymphocytes was assessed in unstimulated PBMCs before the first immunization (pre-imm), 2 weeks after final immunization (post-imm), and the day before CHMI (pre-CHMI). Fold change compared to pre-imm is shown for total memory γδ T cells (a), Vγ9+Vδ2+ (b), Vγ9+Vδ1+ (c), Vγ9Vδ1+ (d), Vγ9+Vδ1Vδ2 (e), and Vγ9Vδ1Vδ2 (f) subfamilies. The frequency of Vγ9Vδ2+ subset is low to undetectable. Within a dose group, the difference from pre-imm was assessed by two-way ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01. Data are geometric mean ± 95% CI.

  6. Extended Data Figure 6: Anti-plasmodial antibody responses in vaccinated volunteers who were immunized with three doses of 5.12 × 104 PfSPZ at 28-day, 14-day, or 5-day intervals. (160 KB)

    Antibodies to PfCSP by ELISA were assessed in sera taken before any immunizations (pre-immunization), two weeks following last immunization (post-immunization) and 10 weeks after last immunization, which was one day before CHMI (pre-CHMI). PfCSP ELISA results are reported as net OD 1.0; the reciprocal serum dilution at which the optical density was 1.0 in post-immunization or pre-CHMI sera minus the OD 1.0 in pre-immunization sera. All values met criteria for positivity. Protected volunteers are represented by yellow circles and unprotected volunteers by grey circles.

  7. Extended Data Figure 7: Development of PfSPZ Vaccine and PfSPZ-CVac in hepatocytes. (396 KB)

    Radiation-attenuated PfSPZ in PfSPZ Vaccine invade hepatocytes and partially develop, but do not replicate. They are metabolically active and non-replicating. Infectious PfSPZ in PfSPZ-CVac invade hepatocytes and fully develop. A single PfSPZ replicates exponentially producing more than 104 merozoites. These merozoites are released into the circulation, and each merozoite can invade a different erythrocyte. Chloroquine prevents complete parasite development within erythrocytes, thereby preventing the development of merozoites that can invade new erythrocytes.

  8. Extended Data Figure 8: Transient parasitaemia following vaccination at 5-day intervals. (186 KB)

    Parasitaemia measured by qPCR over 22 days. The subjects who were protected and not protected against CHMI are in yellow and grey, respectively. The times of PfSPZ inoculations are shown as vertical red lines and the time of last CQ administration as a vertical blue line. CQ was given as 10 mg kg−1 (maximum 620 mg) loading dose on day 0 followed by 5 mg kg−1 (maximum 310 mg) chloroquine base on days 5, 10, and 15.

Extended Data Tables

  1. Extended Data Table 1: Pf-specific T-cell correlates of sterile protection, stratified by group (566 KB)

Supplementary information

PDF files

  1. Supplementary Information (804 KB)

    This file contains Supplementary Figure 1, Supplementary Tables 1-5 and 7-9.

  2. Supplementary Table 6 (1.7 MB)

    Supplementary Table 6 shows logistic regression of peak antibody levels (2 weeks after final immunization) and baseline antibody levels on probability of sterile protection against CHMI, adjusted by dose of PfSPZ-CVac.

Additional data