Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody

Article metrics

Abstract

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Binding kinetics and epitope bins for the human anti-PvDBPII mAb panel.
Fig. 2: Inhibition of the binding of recombinant PvDBPII to the DARC ectodomain.
Fig. 3: Growth inhibition of transgenic P. knowlesi lines expressing PvDBP.
Fig. 4: Inhibition of invasion of reticulocytes by Thai P. vivax clinical isolates.
Fig. 5: Assessment of synergy, additivity and antagonism by anti-PvDBPII human mAb combinations.
Fig. 6: Structural basis for inhibition of PvDBPII by the antibody DB9.

Data availability

Crystallographic data are deposited in the Protein Data Bank with accession number 6R2S. DNA sequences are deposited in GenBank with accession numbers MK75250524. The remaining data that support the findings of this study are available from the corresponding author on request.

References

  1. 1.

    Control and Elimination of Plasmodium vivax Malaria: a Technical Brief (WHO, 2015).

  2. 2.

    Gething, P. W. et al. A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS Negl. Trop. Dis. 6, e1814 (2012).

  3. 3.

    Gruszczyk, J. et al. Transferrin receptor 1 is a reticulocyte-specific receptor for Plasmodium vivax. Science 359, 48–55 (2018).

  4. 4.

    Malleret, B., Renia, L. & Russell, B. The unhealthy attraction of Plasmodium vivax to reticulocytes expressing transferrin receptor 1 (CD71). Int. J. Parasitol. 47, 379–383 (2017).

  5. 5.

    Singh, S. K., Hora, R., Belrhali, H., Chitnis, C. E. & Sharma, A. Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain. Nature 439, 741–744 (2006).

  6. 6.

    Chitnis, C. E. & Sharma, A. Targeting the Plasmodium vivax Duffy-binding protein. Trends Parasitol. 24, 29–34 (2008).

  7. 7.

    Singh, A. P. et al. Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion. Mol. Microbiol. 55, 1925–1934 (2005).

  8. 8.

    Singh, A. P., Puri, S. K. & Chitnis, C. E. Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion. Mol. Biochem. Parasitol. 121, 21–31 (2002).

  9. 9.

    Miller, L. H., Mason, S. J., Clyde, D. F. & McGinniss, M. H. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N. Engl. J. Med. 295, 302–304 (1976).

  10. 10.

    Howes, R. E. et al. Plasmodium vivax transmission in Africa. PLoS Negl. Trop. Dis. 9, e0004222 (2015).

  11. 11.

    Culleton, R. L. et al. Failure to detect Plasmodium vivax in West and Central Africa by PCR species typing. Malar. J. 7, 174 (2008).

  12. 12.

    Draper, S. J. et al. Malaria vaccines: recent advances and new horizons. Cell Host Microbe 24, 43–56 (2018).

  13. 13.

    Batchelor, J. D., Zahm, J. A. & Tolia, N. H. Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC. Nat. Struct. Mol. Biol. 18, 908–914 (2011).

  14. 14.

    Choe, H. et al. Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC). Mol. Microbiol. 55, 1413–1422 (2005).

  15. 15.

    Batchelor, J. D. et al. Red blood cell invasion by Plasmodium vivax: structural basis for DBP engagement of DARC. PLoS Pathog. 10, e1003869 (2014).

  16. 16.

    Hans, D. et al. Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion. Mol. Microbiol. 55, 1423–1434 (2005).

  17. 17.

    VanBuskirk, K. M., Sevova, E. & Adams, J. H. Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition. Proc. Natl Acad. Sci. USA 101, 15754–15759 (2004).

  18. 18.

    Singh, K. et al. Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a phase I trial. NPJ Vaccines 3, 48 (2018).

  19. 19.

    Payne, R. O. et al. Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies. JCI Insight 2, e93683 (2017).

  20. 20.

    De Cassan, S. C. et al. Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein Region II of Plasmodium vivax. Front Immunol. 6, 348 (2015).

  21. 21.

    Moreno, A. et al. Preclinical assessment of the receptor-binding domain of Plasmodium vivax Duffy-binding protein as a vaccine candidate in rhesus macaques. Vaccine 26, 4338–4344 (2008).

  22. 22.

    Bermudez, M., Moreno-Perez, D. A., Arevalo-Pinzon, G., Curtidor, H. & Patarroyo, M. A. Plasmodium vivax in vitro continuous culture: the spoke in the wheel. Malar. J. 17, 301 (2018).

  23. 23.

    Cole-Tobian, J. L. et al. Strain-specific Duffy binding protein antibodies correlate with protection against infection with homologous compared to heterologous Plasmodium vivax strains in Papua New Guinean children. Infect. Immun. 77, 4009–4017 (2009).

  24. 24.

    King, C. L. et al. Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection. Proc. Natl Acad. Sci. USA 105, 8363–8368 (2008).

  25. 25.

    Nicolete, V. C., Frischmann, S., Barbosa, S., King, C. L. & Ferreira, M. U. Naturally acquired binding-inhibitory antibodies to Plasmodium vivax Duffy binding protein and clinical immunity to malaria in rural Amazonians. J. Infect. Dis. 214, 1539–1546 (2016).

  26. 26.

    Chen, E. et al. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy binding protein. Proc. Natl Acad. Sci. USA 113, 6277–6282 (2016).

  27. 27.

    Chootong, P. et al. Mapping epitopes of the Plasmodium vivax Duffy binding protein with naturally acquired inhibitory antibodies. Infect. Immun. 78, 1089–1095 (2010).

  28. 28.

    Ye, J., Ma, N., Madden, T. L. & Ostell, J. M. IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res. 41, W34–W40 (2013).

  29. 29.

    Shakri, A. R., Rizvi, M. M. & Chitnis, C. E. Development of quantitative receptor–ligand binding assay for use as a tool to estimate immune responses against Plasmodium vivax Duffy binding protein Region II. J. Immunoass. Immunochem. 33, 403–413 (2012).

  30. 30.

    Moon, R. W. et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc. Natl Acad. Sci. USA 110, 531–536 (2013).

  31. 31.

    Mohring, F. et al. Rapid and iterative genome editing in the zoonotic malaria parasite Plasmodium falciparum: new tools for P. vivax research. Preprint at https://www.biorxiv.org/content/10.1101/590976v1.full (2019).

  32. 32.

    Adams, J. H. et al. The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63, 141–153 (1990).

  33. 33.

    Gruring, C. et al. Human red blood cell-adapted Plasmodium knowlesi parasites: a new model system for malaria research. Cell Microbiol. 16, 612–620 (2014).

  34. 34.

    Pain, A. et al. The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature 455, 799–803 (2008).

  35. 35.

    Ord, R. L. et al. A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein. Malar. J. 13, 326 (2014).

  36. 36.

    Douglas, A. D. et al. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5. J. Immunol. 192, 245–258 (2014).

  37. 37.

    Saul, A. Kinetic constraints on the development of a malaria vaccine. Parasite Immunol. 9, 1–9 (1987).

  38. 38.

    Williams, A. R. et al. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens. PLoS Pathog. 8, e1002991 (2012).

  39. 39.

    Nobrega de Sousa, T., Carvalho, L. H. & Alves de Brito, C. F. Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development. PLoS ONE 6, e22944 (2011).

  40. 40.

    Tiller, T. et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112–124 (2008).

  41. 41.

    Quan, J. & Tian, J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS ONE 4, e6441 (2009).

  42. 42.

    Wrammert, J. et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671 (2008).

  43. 43.

    Jin, J. et al. Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal ‘C-tag’. Int. J. Parasitol. 47, 435–446 (2017).

  44. 44.

    Ntumngia, F. B. et al. Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies. Infect. Immun. 80, 1203–1208 (2012).

  45. 45.

    Hostetler, J. B. et al. A library of Plasmodium vivax recombinant merozoite proteins reveals new vaccine candidates and protein–protein interactions. PLoS Negl. Trop. Dis. 9, e0004264 (2015).

  46. 46.

    Payne, R. O., Griffin, P. M., McCarthy, J. S. & Draper, S. J. Plasmodium vivax controlled human malaria infection—progress and prospects. Trends Parasitol. 33, 141–150 (2017).

  47. 47.

    Rijal, P. et al. Therapeutic monoclonal antibodies for Ebola virus infection derived from vaccinated humans. Cell Rep. 27, 172–186.e7 (2019).

  48. 48.

    Miura, K. et al. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting Plasmodium falciparum growth, as determined by the in vitro growth inhibition assay. Clin. Vaccin. Immunol. 16, 963–968 (2009).

  49. 49.

    Kennedy, M. C. et al. In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response. Infect. Immun. 70, 6948–6960 (2002).

  50. 50.

    Smolarek, D. et al. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines. Cell Mol. Life Sci. 67, 3371–3387 (2010).

  51. 51.

    Cho, J. S. et al. Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry. Int. J. Parasitol. 46, 31–39 (2016).

  52. 52.

    Russell, B. et al. A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax. Blood 118, e74–e81 (2011).

  53. 53.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  54. 54.

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

  55. 55.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  56. 56.

    Bricogne, G. et al. Buster version 2.10.2 (Global Phasing Limited, 2017).

  57. 57.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

Download references

Acknowledgements

The authors are grateful for the assistance of J. Furze, D. Llewellyn, D. Worth, K. Johnson, J. Barrett and S. Biswas (Jenner Institute, University of Oxford), P. C. Wilson (University of Chicago) for the expression plasmids, O. Fedorov (Structural Genomics Consortium, University of Oxford) for use of the Octet RED384, A. Duckett and C. Banner (University of Oxford) for arranging contracts, Y. Durocher for the HEK293E cells (CNRC-NRC, Canada), A. Rushdi Shakri (ICGEB, India) and C. E. Chitnis (Institute Pasteur, France) for the PvDBPII variant proteins, J. McCarthy (QIMR, Australia) for the HMP013 P. vivax strain PvDBPII sequence, O. Bertrand (INSERM, Paris) for the VHH camelid anti-DARC nanobody, and the VAC051 PvDBPII vaccine clinical trial study volunteers and staff and patients associated with the Shoklo Malaria Research Unit for the P. vivax sample donation (SMRU, Thailand). This work was supported in part by funding from the European Union’s Horizon 2020 Research and Innovation programme under a grant agreement for MultiViVax (number 733073). T.A.R. held a Wellcome Trust Research Training Fellowship (108734/Z/15/Z), N.M.B. is funded by the Wellcome Trust DPhil programme in structural cell biology, J.S.C. held a Singapore International Graduate Award (SINGA), D.G.W.A. held a UK Medical Research Council (MRC) iCASE PhD Studentship (MR/K017632/1), M.K.H. is a Wellcome Trust investigator (101020/Z/13/Z), and S.J.D. is a Jenner investigator, a Lister Institute Research Prize fellow and a Wellcome Trust senior fellow (106917/Z/15/Z). SMRU is part of the Mahidol Oxford Tropical Medicine Research Unit supported by the Wellcome Trust. R.W.M. and F.M. are supported by a UK MRC Career Development Award (MR/M021157/1) jointly funded by the UK MRC and Department for International Development.

Author information

T.A.R., N.M.B., M.K.H. and S.J.D. conceived the study and wrote the manuscript. T.A.R., N.M.B., F.M., J.S.C., V.K., S.F.G., D.G.W.A., G.M.L., S.C.E., S.E.S., D.Q., J.J., J.M.M., R.W.M. and M.K.H. performed the experiments. T.A.R., N.M.B., F.M., J.S.C., D.G.W.A., M.K.H. and S.J.D. performed the data analysis and interpretation. R.O.P., A.M.M., J.J., R.W.M., B.R., L.R. and F.H.N. contributed reagents or materials and facilities.

Correspondence to Matthew K. Higgins or Simon J. Draper.

Ethics declarations

Competing interests

D.G.W.A., M.K.H. and S.J.D. are named inventors on patent applications relating to malaria vaccines, mAbs and/or immunization regimens.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–5 and Supplementary Tables 1–6.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rawlinson, T.A., Barber, N.M., Mohring, F. et al. Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody. Nat Microbiol 4, 1497–1507 (2019) doi:10.1038/s41564-019-0462-1

Download citation

Further reading