Abstract
Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Data to support the findings of study are available from the corresponding author upon request without restrictions. Source data are provided with this paper.
References
World Health Organization. World Malaria Report 2021. WHO, 2021.
Cowman, A. F., Healer, J., Marapana, D. & Marsh, K. Malaria: biology and disease. Cell 167, 610–624 (2016).
Crompton, P. D. et al. Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease. Annu Rev. Immunol. 32, 157–187 (2014).
Laurens, M. B. RTS,S/AS01 vaccine (Mosquirix): an overview. Hum. Vaccin Immunother. 16, 480–489 (2020).
Duffy, P. E. & Patrick Gorres, J. Malaria vaccines since 2000: progress, priorities, products. NPJ Vaccines 5, 48 (2020).
Datoo, M. S. et al. Efficacy and immunogenicity of R21/Matrix-M vaccine against clinical malaria after 2 years’ follow-up in children in Burkina Faso: a phase 1/2b randomised controlled trial. Lancet Infect. Dis. 22, 1728–1736 (2022).
Datoo, M. S. et al. Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial. Lancet 397, 1809–1818 (2021).
Fernandez-Ruiz, D. et al. Liver-resident memory CD8+ T cells form a front-line defense against malaria liver-stage infection. Immunity 45, 889–902 (2016).
Seder, R. A. et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341, 1359–1365 (2013).
Epstein, J. E. et al. Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity. Science 334, 475–480 (2011).
Schofield, L. et al. γ Interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 220, 664–666 (1987).
Lefebvre, M. N. et al. Expeditious recruitment of circulating memory CD8 T cells to the liver facilitates control of malaria. Cell Rep. 37, 109956 (2021).
Tse, S. W., Radtke, A. J., Espinosa, D. A., Cockburn, I. A. & Zavala, F. The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8+ T cells specific for infectious pathogens. J. Infect. Dis. 210, 1508–1516 (2014).
Holz, L. E. et al. CD8+ T cell activation leads to constitutive formation of liver tissue-resident memory T cells that seed a large and flexible niche in the liver. Cell Rep. 25, 68–79 (2018).
Nunes-Cabaco, H., Moita, D. & Prudencio, M. Five decades of clinical assessment of whole-sporozoite malaria vaccines. Front. Immunol. 13, 977472 (2022).
Ishizuka, A. S. et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat. Med. 22, 614–623 (2016).
Jongo, S. A. et al. Immunogenicity and protective efficacy of radiation-attenuated and chemo-attenuated PfSPZ vaccines in Equatoguinean adults. Am. J. Trop. Med. Hyg. 104, 283–293 (2021).
Oneko, M. et al. Safety, immunogenicity and efficacy of PfSPZ vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial. Nat. Med. 27, 1636–1645 (2021).
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).
Ocana-Morgner, C., Mota, M. M. & Rodriguez, A. Malaria blood stage suppression of liver stage immunity by dendritic cells. J. Exp. Med. 197, 143–151 (2003).
Olsen, T. M., Stone, B. C., Chuenchob, V. & Murphy, S. C. Prime-and-trap malaria vaccination to generate protective CD8+ liver-resident memory T cells. J. Immunol. 201, 1984–1993 (2018).
Gola, A. et al. Prime and target immunization protects against liver-stage malaria in mice. Sci. Transl. Med. 10, eaap9128 (2018).
Holz, L. E. et al. Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria. Sci. Immunol. 5, eaaz8035 (2020).
Brennan, P. J., Brigl, M. & Brenner, M. B. Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat. Rev. Immunol. 13, 101–117 (2013).
Heide, J., Vaughan, K. C., Sette, A., Jacobs, T. & Schulze Zur Wiesch, J. Comprehensive Review of human Plasmodium falciparum-specific CD8+ T cell epitopes. Front. Immunol. 10, 397 (2019).
Morita, M. et al. Structure-activity relationship of alpha-galactosylceramides against B16-bearing mice. J. Med. Chem. 38, 2176–2187 (1995).
Hogquist, K. A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994).
Compton, B. J. et al. Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist. Org. Biomol. Chem. 17, 1225–1237 (2019).
Compton, B. J. et al. Synthesis and activity of 6′′-deoxy-6′′-thio-α-GalCer and peptide conjugates. Org. Lett. 17, 5954–5957 (2015).
Hung, J. T. et al. Design and synthesis of galactose-6-OH-modified α-galactosyl ceramide analogues with Th2-biased immune responses. RSC Adv. 4, 47341–47356 (2014).
Chennamadhavuni, D. et al. Dual modifications of α-galactosylceramide synergize to promote activation of human invariant natural killer T cells and stimulate anti-tumor immunity. Cell Chem. Biol. 25, 571–584 (2018).
Christo, S. N. et al. Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity. Nat. Immunol. 22, 1140–1151 (2021).
Carvalho, L. H. et al. IL-4-secreting CD4+ T cells are crucial to the development of CD8+ T-cell responses against malaria liver stages. Nat. Med. 8, 166–170 (2002).
Valencia-Hernandez, A. M. et al. A natural peptide antigen within the Plasmodium ribosomal protein RPL6 confers liver TRM cell-mediated immunity against malaria in mice. Cell Host Microbe 27, 950–962 (2020).
Evrard, M. et al. Sphingosine 1-phosphate receptor 5 (S1PR5) regulates the peripheral retention of tissue-resident lymphocytes. J. Exp. Med. 219, e20210116 (2022).
Grunwitz, C. et al. HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory. Oncoimmunology 8, e1629259 (2019).
Salomon, N. et al. Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer. Cancer Immunol. Immunother. 71, 1975–1988 (2022).
Salomon, N. et al. A liposomal RNA vaccine inducing neoantigen-specific CD4+ T cells augments the antitumor activity of local radiotherapy in mice. Oncoimmunology 9, 1771925 (2020).
Sahin, U. et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 585, 107–112 (2020).
Kranz, L. M. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396–401 (2016).
Mallory, K. L. et al. Messenger RNA expressing PfCSP induces functional, protective immune responses against malaria in mice. NPJ Vaccines 6, 84 (2021).
Godfrey, D. I., Le Nours, J., Andrews, D. M., Uldrich, A. P. & Rossjohn, J. Unconventional T cell targets for cancer immunotherapy. Immunity 48, 453–473 (2018).
Giaccone, G. et al. A phase I study of the natural killer T-cell ligand alpha-galactosylceramide (KRN7000) in patients with solid tumors. Clin. Cancer Res. 8, 3702–3709 (2002).
Tefit, J. N. et al. Efficacy of ABX196, a new NKT agonist, in prophylactic human vaccination. Vaccine 32, 6138–6145 (2014).
Guevara, M. L., Jilesen, Z., Stojdl, D. & Persano, S. Codelivery of mRNA with α-galactosylceramide using a new lipopolyplex formulation induces a strong antitumor response upon intravenous administration. ACS Omega 4, 13015–13026 (2019).
Verbeke, R. et al. Broadening the message: a nanovaccine co-loaded with messenger RNA and α-GalCer induces antitumor immunity through conventional and natural killer T cells. ACS Nano 13, 1655–1669 (2019).
Miyamoto, K., Miyake, S. & Yamamura, T. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413, 531–534 (2001).
Kain, L. et al. The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides. Immunity 41, 543–554 (2014).
Schmidt, N. W. et al. Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria. Proc. Natl Acad. Sci. USA 105, 14017–14022 (2008).
Murphy, S. C., Kas, A., Stone, B. C. & Bevan, M. J. A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations. Proc. Natl Acad. Sci. USA 110, 6055–6060 (2013).
Wilson, N. S. et al. Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat. Immunol. 7, 165–172 (2006).
Chandra, S. et al. A new mouse strain for the analysis of invariant NKT cell function. Nat. Immunol. 16, 799–800 (2015).
Kawabe, T. et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167–178 (1994).
Hildner, K. et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 322, 1097–1100 (2008).
Kennedy, M. K. et al. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J. Exp. Med. 191, 771–780 (2000).
Madsen, L. et al. Mice lacking all conventional MHC class II genes. Proc. Natl Acad. Sci. USA 96, 10338–10343 (1999).
Meijlink, M. A. et al. 6′′-modifed α-GalCer-peptide conjugate vaccine candidates protect against liver-stage malaria. RSC Chem. Biol. 3, 551–560 (2022).
Benedict, M. Q. in The Molecular Biology of Insect Disease Vectors (eds Crampton, J. M. et al.) Ch. 1 (Chapman & Hall, 1997).
Kimura, K. et al. CD8+ T cells specific for a malaria cytoplasmic antigen form clusters around infected hepatocytes and are protective at the liver stage of infection. Infect. Immun. 81, 3825–3834 (2013).
Cockburn, I. A. et al. Dendritic cells and hepatocytes use distinct pathways to process protective antigen from Plasmodium in vivo. PLoS Pathog. 7, e1001318 (2011).
Beattie, L. et al. A transcriptomic network identified in uninfected macrophages responding to inflammation controls intracellular pathogen survival. Cell Host Microbe 14, 357–368 (2013).
Chatzileontiadou, D. S. M., Szeto, C., Jayasinghe, D. & Gras, S. Protein purification and crystallization of HLA-A∗02:01 in complex with SARS-CoV-2 peptides. STAR Protoc. 2, 100635 (2021).
Farrand, K. et al. Using full-spectrum flow cytometry to phenotype memory T and NKT cell subsets with optimized tissue-specific preparation protocols. Curr. Protoc. 2, e482 (2022).
Acknowledgements
We thank the BRF at the Peter Doherty Institute, and the BRU and the Hugh Green Cytometry Centre at the Malaghan Institute of Medical Research for technical support. We thank D. Bowen for support of K.E., D. Godfrey for the CD1d–PBS-44 tetramers and the NIH tetramer core facility for the CD1d–PBS-57 tetramers. This work was supported by funding from the New Zealand Ministry of Business Innovation and Employment (contract RTVU1603 to Victoria University of Wellington) and the New Zealand Health Research Council (contract HRC-20/569 to Victoria University of Wellington and HRC14/1003 Independent Research Organisation Fund to the Malaghan Institute). K.C.Y.P. is supported by the Monash Graduate Scholarship and the Monash University MNHS Faculty International Tuition Scholarship. D.F.R. was supported by the National Health and Medical Research Council of Australia (NHMRC) 1139486, S.G. by NHMRC 1159272, P.B. and K.E. by NHMRC 1146677, W.R.H. by NHMRC 1154457, and W.R.H. and L.E.H. by NHMRC 2012701.
Author information
Authors and Affiliations
Contributions
W.R.H., I.F.H, G.F.P, D.F.-R., L.E.H., D.F.A. and M.G. conceived the idea and designed the outline of the research. A.C., I.A.C., K.Y. and G.I.M. provided sporozoites for infection studies. K.C.Y.P., J.L.N. and J.R. carried out affinity measurements of 2C12 TCR–CD1d–αGCB by surface plasmon resonance. W.R.H., I.F.H., G.F.P., L.E.H. and M.G. wrote the manuscript. J.L.N., S.L.D., S.T.S.C, R.J.A., B.J.C. A.J.M. O.K.B. and J.J.M. wrote Methods sections. All authors contributed to reviewing and editing the manuscript and to relevant discussions. M.G., K.J.F., O.K.B., J.C.M., M.M., L.E.H., J.J.M., Y.C.C. and Z.G. vaccinated mice, analyzed T cell responses and examined protection. S.L.D., M.G., J.J.M., S.T.S.C, R.J.A., B.J.C. and A.M. prepared vaccines and vaccine components. S.G. produced Kb tetramers. C.X. produced CD1d–PBS-44 tetramers. K.E. and P.B. contributed to analysis of liver DC.
Corresponding authors
Ethics declarations
Competing interests
M.G., L.E.H., R.J.A., B.J.C., A.J.M. I.F.H., W.R.H. and G.F.P. are inventors on a patent application (WO2023121483A1) submitted by Victoria University of Wellington subsidiary Victoria Link Limited that covers the production of tissue-resident memory T cells with mRNA vaccines.
Peer review
Peer review information
Nature Immunology thanks Rafick Sekaly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Jamie D. K. Wilson, in collaboration with the Nature Immunology team.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Vaccine-induced activation of splenic NKT cells.
Male B6 mice were transferred OT-I.CD45.1 cells one day before vaccination with mOVA vaccine alone, or mOVA with 80 pmol of indicated adjuvants. Data are combined from two independent experiments, giving n = 10 mice per group with the exception of the mOVA + αGCB group which contains 9 mice. a, Example flow cytometry plots showing expression of the TCR on splenic NKT cells at day 28. Gating shown in Supplementary data Fig. 4. b, Examples of CD69 (left) and PD-1 (right) expression on splenic NKT cells. c, Percentage TCRβ+ CD1d-tetramer+ spleen cells as gated in (a). Data are displayed as mean ± s.e.m. and individual mice (circles) and compared to the mOVA + αGC group by one-way ANOVA analysis with Tukey’s multiple comparison post-test. d, Geometric mean fluorescence intensity of PD-1 on NKT cells. Data are displayed as mean ± s.e.m. from two independent experiments and compared to the mOVA + αGC group by one-way ANOVA analysis with Tukey’s multiple comparison post-test. ****P < 0.0001.
Extended Data Fig. 2 Type I NKT 2C12 TCR binding affinity for mouse CD1d-αGCB.
Affinity measurement of soluble mouse type I NKT 2C12 TCR to mCD1d-αGCB using surface plasmon resonance (SPR). a, Equilibrium curves representative of one experiment performed in duplicate. b, Kd values are derived from duplicate runs from n = 3 independent experiments. Error bars on lower graph show mean ± SD.
Extended Data Fig. 3 Effect of vaccination on expression of costimulatory molecules on liver DC.
Female B6 mice were vaccinated with mOVA vaccine alone or mOVA with adjuvants αGC or αGCB. Livers were harvested 24 h later and DC (CD11c+ MHC II+ cells) assessed for relative expression of CD80 (a) and CD86 (b). Data are expressed as percent of maximum (% of max) determined by dividing the GMFI of each sample by the maximum mean GMFI of the highest mean of all groups from each experiment. Each circle depicts the values of an individual mouse (n = 10 biologically distinct samples combined from two independent experiments). Lines show mean ± s.e.m. Data (a, b) were compared by a one-way ANOVA with Tukey’s multiple comparison post-test (a, b). ****P < 0.0001.
Extended Data Fig. 4 The role of cytokines in responses to mRNA vaccination.
a, Male B6 mice were transferred OT-I.CD45.1 cells one day before vaccination with mOVA + αGCB. Groups of these mice were injected with mAb specific for blocking either IL-12p35, IL-15, GM-CSF or IFN-γR1, or with an isotype control mAb on days 0, 1 and 3 and then left to day 30 before OT-I memory T cell subsets were quantified in the liver. Gating parameters for OT-I cells are described in Supplementary data Fig. 1. Data are expressed as mean cell count ± s.e.m. for each subset. Individual values for each mouse are shown in Supplementary Fig. 8a. b, Female B6 mice or IL-15−/− mice were transferred OT-I.CD45.1 cells one day before vaccination with mOVA + αGCB., and 36 days later OT-I memory T cell subsets were quantified in the liver. Mean cell count ± s.e.m. for each subset shown. Individual values for each mouse are shown in Supplementary Fig. 8b. c, Male B6 mice were OT-I.CD45.1 cells one day before vaccination mOVA + αGCB. Groups of these mice were injected with specific mAb for blocking IFNαR1 or an isotype control on days 0, 1 and 3 and then left to day 30 before OT-I memory T cell subsets were quantified in the liver. Mean cell count ± s.e.m. for each subset shown. Individual values for each mouse are shown in Supplementary data 8c. Data in a were log-transformed and compared to the isotype group by one-way ANOVA with Tukey’s multiple comparison post-test. Data in b and c were log-transformed and compared by two-sided unpaired Student’s t tests. Two independent experiments were performed for each panel (a,b, n = 10 mice per group; c, n = 7 mice per group). ****P < 0.0001. Bars indicating significance are coloured to correspond to each memory T cell subset.
Extended Data Fig. 5 Assessment of total anti-OVA IgG in sera 3 weeks following a prime-boost schedule.
Male B6 mice were vaccinated with mOVA vaccine alone (containing 5 µg mRNA), or mOVA adjuvanted with αGC or αGCB, on days 0 and 21, with sera collected 3 weeks after the boost analysed for total anti-OVA IgG by ELISA. Two independent experiments were combined to give 10 mice per group with the exception of the αGC group which contains 11 mice. Geometric mean ± 95 % confidence intervals for EC50 values are shown. Data were log-transformed and compared by one-way ANOVA with Tukey’s multiple comparison post-test. ****P < 0.0001.
Supplementary information
Supplementary Information
Supplementary Figs. 1–14 and Table 1.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Fig. 7
Statistical source data.
Source Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data.
Source Data Extended Data Fig. 2
Statistical source data.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig. 5
Statistical source data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ganley, M., Holz, L.E., Minnell, J.J. et al. mRNA vaccine against malaria tailored for liver-resident memory T cells. Nat Immunol 24, 1487–1498 (2023). https://doi.org/10.1038/s41590-023-01562-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41590-023-01562-6
This article is cited by
-
Leveraging malaria vaccines and mRNA technology to tackle the global inequity in pharmaceutical research and production towards disease elimination
Malaria Journal (2024)
-
Warum die Regeneration von immunologischer Toleranz durch Impfen schwierig ist
Zeitschrift für Rheumatologie (2024)
-
Alt-RNAtive vaccines elicit anti-malarial TRM cells
Nature Immunology (2023)