Abstract
Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year1. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum2, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150–200 genes per parasite genome3 that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.
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Change history
14 February 2018
Change history: Please see accompanying Corrigendum (http://doi.org/10.1038/nature25498). In Extended Data Fig. 1, the flow cytometry panel showing uninfected erythrocytes stained with LILRB5-Fc was inadvertently duplicated
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Acknowledgements
We thank T. Mitamura for discussions, K. Saito for mass spectrometry analysis, M. Matsumoto and S. Matsuoka for technical assistance, the Thai and Tanzanian donors and the Japanese Red Cross Society for providing human erythrocytes and human plasma. This work was partly supported by the Japanese Initiative for Progress of Research on Infectious Disease for Global Epidemic from the Japan Agency for Medical Research and Development (AMED) (H.A.), the Platform Project for Supporting Drug Discovery and Life Science Research from AMED (J.T.), JSPS KAKENHI grant numbers JP16K08839 (K.H.), JP16H05195 (T.Su.), JP15K08531 (M.K.), MEXT KAKENHI grant numbers JP26117714 (H.A.), JP23117008 (T.T.), JP24115005 (H.A.), the Senri Life Science Foundation (K.H.), the Kato Memorial Bioscience Foundation (K.H.), the Danish Council for Independent Research grants 1333-00220 (C.W.W.) and 4004-00624B (T.L.), The Lundbeck Foundation (T.L.) and the United States National Institutes of Health (NIH R01HL130678, T.L.). F.S was supported by the Taniguchi Memorial Fellowship program.
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Contributions
F.S. and K.H. performed most of the binding and functional experiments, and analysed and discussed the data. T.Sa. performed limiting dilution experiments and assisted with ligand identification. C.W.W. performed binding and serological analyses in Tanzania. J.L. assisted with experiments in Tanzania. T.A. performed CD spectral analysis. K.S. prepared recombinant plasmids. N.M.Q.P. assisted with P. falciparum culture and cloning. S.It. assisted with P. falciparum culture. S.Iw. assisted with transgenic parasite experiments and provided Thai P. falciparum isolates. E.T. prepared wheat germ cell-free proteins. T.T. designed wheat germ cell-free protein expression system and discussed the data. M.K. assisted with functional experiments. T.Su. assisted with ligand identification. M.C. assisted with LILR analyses. J.T. assisted with protein experiments and discussed the data. T.L. assisted with experiments in Tanzania and discussed the data. T.H. assisted with P. falciparum experimental design and discussed the data. H.A. designed the study and analysed the data. All authors contributed to the writing of the manuscript.
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Competing financial interests: Osaka university has filed a provisional patent application that covers the use of LILRB1-binding RIFINs as a possible target for malaria vaccine. F.S., K.H. and H.A. are listed as inventors.
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Extended data figures and tables
Extended Data Figure 1 Binding of Fc fusion proteins of inhibitory receptors to IEs.
Erythrocytes infected with 3D7 (3D7 IEs), P. falciparum obtained from patient 1 (Patient 1 IEs), or uninfected erythrocytes were stained with Fc fusion proteins of inhibitory receptors. As a control, IEs were stained with APC-labelled anti-human IgG Fc Ab alone (Control). FSC, forward-scattered light. The experiments were replicated twice.
Extended Data Figure 2 Variability and stability of LILRB1 binding to IEs and LILRB1 reporter activity.
a, LILRB1 binding to schizont-stage P. falciparum-infected erythrocytes from patients with malaria in Fig. 1a. b, LILRB1 binding to P. falciparum-infected erythrocytes derived from patient 6 in Fig. 1b at the ring, mid-trophozoite and schizont stages. c, LILRB1 binding to schizont-stage P. falciparum-infected erythrocytes from the laboratory strains CDC1, K1, FCR3 and Dd2 shown in Fig. 1c. d, GFP expression in LILRB1-expressing reporter cells upon stimulation with P. falciparum-infected erythrocytes in Fig. 3c. Data represent the mean ± s.d. of three independent experiments. e, Proportions of LILRB1–Fc- binding erythrocytes infected with clone 3D7-F2 were analysed during 5 weeks of culture. The experiment was performed once.
Extended Data Figure 3 Identification of the LILRB1 ligand.
a, Diagram of LILRB1 ligand identification. A putative LILRB1 ligand was immunoprecipitated from IE ghosts using an LILRB1–Fc fusion protein and was identified using mass spectrometry analysis. b, Mass spectrometry of LILRB1–Fc immunoprecipitates. The observed m/z values of b-ions (red) and y-ions (blue) in the MS/MS spectra of the peptide FHEYDER present in reversed phase high performance liquid chromatography (RP-HPLC) fractions of trypsin digests of LILRB1 precipitates from IEs infected with F2 clones. The experiments were replicated twice. c, The observed m/z values of b-ions and y-ions in the MS/MS spectra of the peptide FHEYDER present in RP-HPLC fractions of trypsin digests of LILRB1 precipitates. The predicted m/z values are shown for comparison. The differences between the m/z values for observed ions and the predicted values are shown.
Extended Data Figure 4 Screening of RIFINs that bound to the LILRB1–Fc or LAIR1–Fc fusion protein.
a, IEs of 3D7 carrying RIFIN transgenes were stained with the LILRB1–Fc fusion protein. RIFIN transgenes are indicated. Red and shaded-grey histograms indicate staining with LILRB1–Fc and control–Fc fusion proteins, respectively. b, IEs of 3D7 carrying RIFIN transgenes were stained with the LAIR1–Fc fusion protein. RIFIN transgenes are indicated. Red and shaded-grey histograms indicate staining with LAIR1–Fc and control–Fc fusion proteins, respectively. The presence of the FHEYDER sequence in each RIFIN is indicated in the figure. Representative data from independent analyses are shown. Therefore, the proportions of IEs bound to Fc fusion proteins and the levels of Fc fusion protein binding IEs may not be comparable among different RIFINs. All experiments were replicated twice.
Extended Data Figure 5 Expression of RIFINs in transgenic malaria parasites.
a, The rif transgene transcript levels normalized to the internal control gene. The average of RIFIN #1 transcript levels was defined as 1. Data represent the mean ± s.d. (n = 3 technically independent samples). RIFIN #6 expression was lowest among the transgenes. However, cell surface expression of RIFIN #6 was detected using a mutated LAIR1–Fc fusion protein (Extended Data Fig. 8a), indicating that all the transgenes were sufficiently expressed at the transcript level. b, Western blot analysis of the expression of transfected C-terminally His-tagged RIFINs transfected into malaria parasites using an anti-His-tag monoclonal antibody. The His-tagged RIFINs were detected at approximately equal levels (Supplementary Data). The expected molecular masses are 31.7 (RIFIN #1), 32.9 (RIFIN #2) and 34.8 (RIFIN #5) kDa. The experiment was performed once. c, P. falciparum-infected erythrocytes expressing C-terminally His-tagged RIFIN transgenes (RIFIN #1, PF3D7_1254800; RIFIN #2, PF3D7_0223100; and RIFIN #5, PF3D7_1254200) were stained with LILRB1–Fc (red) and control–Fc (shaded grey). The experiments were replicated at least twice.
Extended Data Figure 6 Recombinant RIFINs.
a, Binding of recombinant RIFINs to LILRB1 produced using a wheat germ cell-free protein expression system. 293T cells expressing transfected LILRB1 or LILRA2 were stained with recombinant His-tagged RIFINs that were produced using a wheat germ cell-free protein expression system. LILRA2 is an activating counterpart of LILRB1 and was used as a control. Red and blue histograms indicate binding of LILRB1+ RIFIN #1 and LILRB1− RIFIN #5, respectively. The shaded-grey histogram represents an unstained control. The experiments were replicated at least twice. b, Production of recombinant RIFINs in E. coli. N-terminally His-tagged variable regions of RIFINs were expressed in E. coli and purified using TALON metal-affinity chromatography. Recombinant RIFINs were analysed using SDS–PAGE and Oriole staining. LILRB1+ RIFIN #1 and LILRB1− RIFIN #5 are shown on the right and left, respectively. The experiments were replicated at least twice. c, CD spectra of recombinant RIFINs. Refolded and purified recombinant RIFIN #1 and #5 were subjected to CD spectral analysis. The spectra are shown as the mean residue ellipticity after subtracting the solvent background. RIFINs #1 and #5 exhibited CD spectra typical of well-folded proteins with α-helix (208 nm + 222 nm) and β-sheet (215 nm) structures. Prediction of the secondary structures of each RIFIN using the BeStSel server (http://bestsel.elte.hu/index.php) yielded α/β values of approximately 30%/10% and 30%/20% for RIFINs #1 and #5, respectively. The experiment was performed once.
Extended Data Figure 7 LILRB1-binding RIFIN did not bind to other LILRs.
a, The sequence encoding the variable region of LILRB1+ RIFIN #1 was transfected into 293T cells, and the transfectants were stained with LILR–Fc fusion proteins. The levels of LILR–Fc binding are indicated as mean fluorescence intensities (MFIs). Control indicates fluorescence of cells reacted only with the secondary antibody. The experiment was performed once. b, c, Binding of LILR–Fc fusion proteins to IEs. RIFIN #1- and RIFIN #5-transgenic IEs were stained with 11 LILR–Fc fusion proteins and analysed using flow cytometry. Control indicates fluorescence of cells reacted only with the secondary antibody. The proportions of IEs stained with LILR–Fc fusion proteins are shown. The experiment in b was replicated twice and experiment in c was performed once.
Extended Data Figure 8 Binding of wild-type and mutated LAIR1 to IEs.
a, IEs from RIFIN-transgenic parasites (RIFIN #1, PF3D7_1254800; RIFIN #2, PF3D7_0223100; RIFIN #3, PF3D7_0500400; RIFIN #4, PF3D7_1000500; RIFIN #5, PF3D7_1254200; RIFIN #6, PF3D7_1400600; RIFIN #7, PF3D7_1040300; and RIFIN #8, PF3D7_1101100) were stained with wild-type LAIR1–Fc (wtLAIR1–Fc, red), mutated LAIR1–Fc (muLAIR1–Fc, blue), and control–Fc (shaded-grey histogram) fusion proteins. The experiments were replicated twice. b, LAIR1–Fc bound to erythrocytes infected with P. falciparum derived from Thai patients with malaria. Schizont-stage erythrocytes infected with P. falciparum from patients with malaria and uninfected erythrocytes were stained with LAIR1–Fc (red dot) and control–Fc (black dot) fusion proteins, followed by Vybrant Green. Percentages of LAIR1-ligand-expressing IEs are shown. The experiments were replicated at least twice. c, Patterns of LILRB1 and LAIR1 binding to IEs derived from Thai patients with malaria. Schizont-stage IEs derived from Thai patients with malaria were stained with LAIR1–Fc (vertical) and LILRB1–Fc (horizontal) fusion proteins, followed by Vybrant Green. Vybrant Green-positive cells were analysed. The percentages of LAIR1-ligand single-positive, LILRB1-ligand single-positive and LAIR- and LILRB1-ligand double-positive IEs are shown. The experiments were replicated twice.
Extended Data Figure 9 Functional analysis of cells expressing LAIR1 and LILRB1.
a–c, Erythrocytes infected with LAIR1–Fc-binding parasites (RIFIN #8 transgenic parasites (a), parasites from Thai patient 1 (b), LAIR1–Fc-binding parasites enriched by cell sorting from Thai patient isolate 4 (LAIR1-L enriched patient 4, c)) or erythrocytes infected with parasites that did not bind LAIR1-Fc (RIFIN #3 transgenic parasites (a), parasites from Thai malaria patient 3 (b, c)) were prepared (left). LAIR1 reporter cells were co-cultured with these IEs and expression levels in reporter cells were analysed using flow cytometry (right). Immobilized collagen IV served as a positive control for LAIR1 reporter activation. Proportions of GFP-expressing cells are shown as the mean ± s.d. (n = 3 biologically independent samples, one-way ANOVA with Tukey’s post hoc test). d, LILRB1 expressed by the NK cell line NKL. e, Flag-tagged RIFINs were expressed by K562 cells stably transfected with LILRB1+ RIFIN #1 or LILRB1− RIFIN #5. f, RIFIN-expressing K562 cells or parental K562 cells were used as targets for NKL. Data represent the mean ± s.d. (n = 3 technically independent samples). *P < 0.05, two-way ANOVA with Tukey’s post hoc test. The experiments in d and e were replicated at least twice.
Extended Data Figure 10 Age dependence of the antibody response to LILRB1+ and LILRB1− RIFINs.
Plasma IgG positivity for the recombinant proteins comprising the variable regions of LILRB1+ RIFIN #1 and LILRB1− RIFIN #5 as well as GLURP_R2 in 222 Tanzanian individuals divided into age groups. Error bars represent 95% confidence interval. P values were calculated using logistic regression comparing per cent responders among children aged 0–1 years to children of the other age groups.
Supplementary information
Supplementary Information
This file contains original gel images, gating strategy for flow cytometry analysis and sequence chromatogram of cloned rif genes. (PDF 16369 kb)
Supplementary Table 1
This file contains information on peptides identified by mass spectrometry analyses. (XLSX 13 kb)
Supplementary Table 2
This file contains information on 33 different rif genes used for generating RIFIN-transgenic parasites. (XLSX 37 kb)
Supplementary Table 3
This file contains information on primers used to clone rif genes. (XLSX 33 kb)
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Saito, F., Hirayasu, K., Satoh, T. et al. Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors. Nature 552, 101–105 (2017). https://doi.org/10.1038/nature24994
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DOI: https://doi.org/10.1038/nature24994
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