Abstract
Trypanosomes are protozoan parasites that cause infectious diseases, including African trypanosomiasis (sleeping sickness) in humans and nagana in economically important livestock1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms—including antigenic variation3—that present an apparently insurmountable barrier to vaccination. Here we show, using a systematic genome-led vaccinology approach and a mouse model of Trypanosoma vivax infection4, that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell-surface protein that is localized to the flagellum membrane (which we term ‘invariant flagellum antigen from T. vivax’) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to this protein could induce sterile protection and revealed several mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained socioeconomic development in countries in sub-Saharan Africa5, and provides evidence that highly protective vaccines against trypanosome infections can be achieved.
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Data availability
Annotated T. vivax genome data were obtained from TriTrypDB (https://tritrypdb.org). All data generated or analysed during this study are included in this Article, and/or are available from the corresponding author (G.J.W., who can also be contacted at gavin.wright@york.ac.uk) on reasonable request. Source data are provided with this paper.
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Acknowledgements
This research was funded by the Wellcome Trust (grant 206194) and BBSRC (grant BB/S001980/1). A.R.-R. was supported by a PhD studentship funded by FONDECYT-CONCYTEC, the National Council of Science, Technology and Innovation from Peru (grant contract number 001-2016-FONDECYT). We are indebted to Institut Pasteur for providing us with the T. vivax luciferase line. We thank Sanger Institute animal technicians for their support with animal work, N. Karp for advice on experimental design, C. Mackenzie for preliminary work on anti-IFX monoclonal antibodies, E. Coomber for help with hybridoma tissue culture, and M. Carrington, L. Morrison and T. Rowan for helpful discussions.
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D.A. and G.J.W. designed the study. D.A. prepared proteins, and performed immunizations and parasite challenges with help from S.C., C.B., H.O. and K.H. C.C. selected hybridomas, cloned the 8E12 monoclonal antibody, and did the isotype switching, mutagenesis and purification. D.A.G. performed immunogold labelling. C.T. generated hybridomas. F.G. and M.K. performed SPR experiments and analysis. A.R.-R., C.W.D. and A.P.J. provided and analysed T. vivax genome sequences and provided sera from infected cattle. D.A. and G.J.W. prepared the manuscript with comments from all co-authors.
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D.A. and G.J.W. are named inventors on two UK patent applications relating to this research.
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Extended data figures and tables
Extended Data Fig. 1 Trypanosoma vivax vaccine candidate antigens, organization of the protection screen and antibody titres.
a, Vaccine candidates were expressed as soluble recombinant proteins in HEK293 cells, purified and resolved by SDS–PAGE to determine protein integrity and purity. For uncropped gel images, see Supplementary Fig. 1. b, Mice were vaccinated with a protein-in-alum formulation using a prime and two-boost regime and rested before challenge with the luciferase-expressing T. vivax parasite line and parasitaemia quantified using bioluminescent imaging. Vaccine candidates were tested in cohorts containing two control cages that were infected first and last to ensure any effect on the reduction of parasite multiplication was not confounded by the loss of parasite virulence. c, Parasite multiplication was identical in mice treated with adjuvant alone compared to naive mice. A group of five mice were immunized three times with alum alone (filled circles) and rested for six weeks and the infection with bioluminescent T. vivax was compared to naive mice (open circles). Data points represent individual mice; grey shading indicates background bioluminescence. d, Serum dilutions for the half-maximal responses for each antigen. Data points are individual mice and bars represent mean ± s.d.; n = 5. ND, not determined. Grey stars indicate no detectable response.
Extended Data Fig. 2 Summary of systematic genome-led reverse vaccinology screen that identified subunit vaccine candidates for T. vivax.
Bioluminescence is used as a proxy for parasitaemia and is shown for each mouse in the indicated days post-challenge. Candidates are identified using their ‘V number’ and organized into their screening cohorts. The two cages of adjuvant-only controls for each cohort are highlighted in grey. The majority of candidates had no effect on the ascending phase of parasitaemia, and are left unshaded. Three candidates (V2, V8 and V31) that had a statistically significant effect on infection are highlighted in pale yellow, and the candidate that elicited strong protection (V23) is highlighted in pale green. Data points are bioluminescence readings from individual mice. The occasional reductions in parasitaemia followed by rebound after day 8 are likely to be due to protective anti-VSG responses and selection of an antigenically distinct variant.
Extended Data Fig. 3 Replication of strong protective effects in a larger group of mice vaccinated with candidate V23 (IFX).
a, Quantification of replicate vaccinations and T. vivax infections with four antigens showing protective effects in the initial screen. V23 and V31 vaccinations replicated, but not V2 and V8. Parasitaemia was quantified on day six using bioluminescence; data points represent individual mice and bars indicate mean ± s.d. ns, not significant, ****P ≤ 0.0001 two-tailed t-test; grey shading indicates background bioluminescence thresholds. b, Fifteen mice were immunized with purified soluble IFX–V23 recombinant protein adjuvanted in alum and challenged with transgenic luciferase-expressing T. vivax. Parasitaemia was quantified on the indicated days after parasite challenge using bioluminescence; controls are a cohort of 15 mice treated with adjuvant only. Ten out of the 15 mice were protected until at least day 170. Data points represent individual mice and grey shading indicates bioluminescence thresholds of uninfected mice. Where mice had to be euthanized for health reasons thought to be unrelated to the infection, this is indicated by a cross.
Extended Data Fig. 4 Parasites do not detectably persist in the organs, adipose tissue or dermis of mice vaccinated with candidate V23 (IFX) challenged with T. vivax.
An IFX–V23-immunized mouse was challenged with luciferase-expressing transgenic T. vivax parasites and protection from infection relative to controls was established. The mouse was rested and, nine months later, injected with luciferin to detect residual parasites using bioluminescence. No bioluminescent signals above background were detected in vaccinated mice either when the whole mouse was imaged, or within the dissected organs, adipose deposits and dermis of IFX–V23-immunized mouse (top panels). An unimmunized mouse was used 8 days after parasite challenge as a positive control (bottom panels).
Extended Data Fig. 5 IFX staining is specific to T. vivax and concentrated at the boundary of the flagellum–cell body contact.
a, Immunogold electron microscopy using an anti-IFX mouse monoclonal antibody localized IFX to clusters along the length of the flagellum in mid-sagittal sections (white arrows and bars). b, Enlarged view of the box in a, showing IFX located between the flagellum and cell membranes. c, Anti-IFX particle staining density was quantified along the membrane interface of the ventral flagellum–cell body (IVC), dorsal flagellum (DF) and cell body area on sagittal and transverse sections. Individual data points are shown; bars represent means. d, e, Control electron micrographs of T. vivax parasites stained with an isotype-matched control mouse IgG1 antibody (d) or goat anti-mouse coated gold particles alone (e), showing no accumulation of gold particles. f, Trypanosoma congolense parasites were stained with anti-IFX rabbit polyclonal sera (top panels) or control preimmune sera (bottom panels) followed by fluorescently conjugated anti-rabbit secondary (red) and counterstained with DAPI (blue). No staining of the parasites was observed demonstrating antibody specificity. Flag., flagellum; fm, flagellar membrane; cm, parasite cell membrane. Scale bars, 100 nm (a, b), 150 nm (d, e), 8 μm (f). Representative images of at least two independent experiments are shown.
Extended Data Fig. 6 Depletion of CD4- and CD8-positive T lymphocytes and natural killer cells in IFX-vaccinated mice before parasite challenge do not affect IFX-mediated protective efficacy.
Groups of mice were vaccinated with IFX, rested and then either natural killer (NK) cells (n = 4) or CD4- (n = 5) or CD8-positive (n = 4) T lymphocytes were depleted using lineage-specific monoclonal antibodies (antibody clone names indicated) before challenging with luciferase-expressing T. vivax parasites. Cell-depleted mice showed no significant difference to control mice treated with an isotype-matched control antibody (LTF-2). The virulence of parasites was confirmed by showing robust infections in naive mice in the same experiment. Parasitaemia was quantified on day 5 using bioluminescence, grey shading indicates bioluminescence thresholds of uninfected mice; data points represent individual mice and bars indicate mean ± s.d.
Extended Data Fig. 7 Identification of the binding epitopes and affinities of a panel of mouse monoclonal antibodies recognizing T. vivax IFX.
a, Schematic showing the N- and C-terminal boundaries of four fragments of the IFX ectodomain. b, Identification of the epitope locations for the anti-IFX monoclonal antibodies. The entire ectodomain (EE) (n = 5) and derived fragments (1 to 4) (n = 3) were expressed as enzymatically biotinylated soluble recombinant proteins in HEK293 cells, immobilized on streptavidin-coated microtitre plates, and the binding of each of the anti-IFX monoclonal antibodies was quantified by ELISA. The hybridoma secreting monoclonal antibody 8F10 was not successfully cloned and therefore not further investigated. Bars represent mean ± s.d. c, Schematic of the antibody binding data to the IFX ectodomain fragments, showing the approximate locations of the antibody epitopes. d, Quantification of the equilibrium binding affinity of the anti-IFX monoclonal antibodies by SPR. Five of the anti-IFX monoclonal antibodies were chemically biotinylated and immobilized on a streptavidin-coated senor chip and the binding to serial dilutions of purified soluble IFX ectodomains was measured. The binding affinity for each of the antibodies at equilibrium (KD) was calculated by fitting the binding data (inset) to a simple 1:1 binding isotherm. KD values are mean ± s.d. using seven analyte dilutions from one experiment. There was no simple positive correlation between the antibody binding affinity and protective efficacy. Antibody affinities were plotted against percentage parasite inhibition in passive transfer experiments at day 5 after infection (mean ± s.d.; n = 5).
Extended Data Fig. 8 Recombinant antibody cloning, isotype switching and mutation of antibody effector recruitment sites of the anti-IFX 8E12 hybridoma.
a, The rearranged variable light and heavy regions of the anti-IFX 8E12 monoclonal antibody were amplified and assembled by fusion PCR using a ‘joining’ fragment before being subcloned into a mammalian protein expression plasmid containing the mouse IgG2a heavy chain. Twelve of fifteen colonies expressed functional anti-IFX antibodies and three selected clones contained identical VH and VL sequences. The 8E12–IgG2a antibody was produced by transfection of HEK293 cells. For uncropped gel images see Supplementary Fig. 1. b, The binding affinity of the 8E12 monoclonal antibody for IFX is unaffected after isotype switching. The biophysical binding parameters of the 8E12 monoclonal antibody for IFX were determined by SPR as both the hybridoma-expressed IgG1 (left) and recombinant IgG2a (right). Serial dilutions of the purified complete ectodomain of IFX were injected for two minutes over the biotinylated antibodies immobilized on a streptavidin-coated sensor chip and left to dissociate. Equilibrium binding constants were calculated by fitting the binding data to a Langmuir binding isotherm and found to be essentially equivalent. c, Mutation of the C1q and FcR recruitment sites on the 8E12–IgG2a heavy chain. The specified mutations that are known to abrogate binding to either C1q or FcR were made on the recombinant 8E12–IgG2a plasmid using site-directed mutagenesis. Mutations were made individually (ΔC1q and ΔFcR) and together (ΔC1qΔFcR). Each of the three mutant antibodies were expressed, purified and IFX-binding activity normalized to the parent 8E12–IgG2a and 8E12–IgG1 by ELISA.
Extended Data Fig. 9 The anti-IFX 8E12–IgG2a monoclonal antibody with abrogated immune effector recruitment sites reveals highly potent protection due to several mechanisms of immunological protection, including a major role for complement.
a, Groups of five mice were injected three times intravenously with the indicated doses of purified anti-IFX 8E12–IgG2a monoclonal antibody and challenged with the luciferase-expressing transgenic T. vivax parasites. Control is an isotype-matched mouse IgG2a monoclonal antibody. A cross indicates where a single mouse had to be removed from the study on day 16 for health reasons thought to be unrelated to the infection. b, Groups of five mice were administered three times intravenously with either 50 μg (left) or 100 μg (right) of purified anti-IFX 8E12–IgG2a monoclonal antibody containing mutations in immune effector recruitment binding sites and challenged with luciferase-expressing transgenic T. vivax parasites. Mutations prevented binding to C1q (ΔC1q), FcRs (ΔFcR) or both (ΔC1qΔFcR) and were compared to non-mutated 8E12–IgG2a, 8E12–IgG1 and both isotype-matched IgG2a and IgG1 controls. In all panels, data points represent individual mice and grey shading indicates bioluminescence thresholds of uninfected mice; dashed lines indicate survival within each group. Reductions in parasitaemia followed by rebounds after day 8 post-infection are likely to be due to the development of protective host antibody responses directed to the dominant VSG within the parasite population and selection of an antigenically distinct variant. One of two independent experiments with very similar outcomes is shown.
Extended Data Fig. 10 IFX adjuvanted in Quil-A and delivered subcutaneously induces consistent and isotype-balanced anti-IFX titres that are highly protective.
a, Groups of five mice were immunized with the purified ectodomain of IFX adjuvanted in alum, Quil-A and montanide ISA 201 VG (mont.) using a prime and two-boost regime either intraperitoneally (alum i.p.) or subcutaneously (Quil-A and montanide s.c.). Half-maximal anti-IFX titres were determined by ELISA. Bars are mean ± s.d. IFX adjuvanted with Quil-A administered subcutaneously were able to elicit anti-IFX antibody titres that were as high as those elicited by IFX adjuvanted with alum delivered intraperitoneally. b, Quantification of different anti-IFX antibody isotypes elicited by the different adjuvants. IFX and Quil-A were able to induce a larger proportion of IgG2 isotype subclasses. Data points represent individual mice (n = 5) and bars are mean ± s.d. c, Increased protection to T. vivax challenge using Quil-A in a protein-in-adjuvant vaccine formulation. Fourteen mice were immunized subcutaneously with purified soluble IFX recombinant protein adjuvanted in Quil-A and challenged with transgenic luciferase-expressing T. vivax. Parasitaemia was quantified on the indicated days after parasite challenge using bioluminescence; controls are a cohort of 14 mice treated with adjuvant only. Data points represent individual mice and grey shading indicates bioluminescence thresholds of uninfected mice. Crosses indicate where individuals had to be removed from the study for health reasons thought to be unrelated to the infection. The smaller bioluminescence peaks in four mice corresponding to high bioluminescent readings between days 16 and 24 were caused by bleed-through of bioluminescence signal from the mouse that eventually succumbed to infection.
Supplementary information
Supplementary Figure 1
Original gels corresponding to Extended Data Figure 1a and Extended Data Figure 8a.
Supplementary Table 1
A table of the Trypanosoma vivax antigens tested in this study.
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Autheman, D., Crosnier, C., Clare, S. et al. An invariant Trypanosoma vivax vaccine antigen induces protective immunity. Nature 595, 96–100 (2021). https://doi.org/10.1038/s41586-021-03597-x
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DOI: https://doi.org/10.1038/s41586-021-03597-x
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