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The Anopheles-midgut APN1 structure reveals a new malaria transmission–blocking vaccine epitope

Nature Structural & Molecular Biology volume 22, pages 532–539 (2015)Cite this article

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Abstract

Mosquito-based malaria transmission–blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV.

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Figure 1: Overview of the AnAPN1 structure.
Figure 2: Structural comparison of AnAPN1 with the open and closed forms of ERAP1.
Figure 3: Catalytic site.
Figure 4: Proposed AnAPN1 dimer.
Figure 5: Enzymatic characterization of AnAPN1.
Figure 6: Anti-AnAPN1 mAb 4H5B7 recognizes AnAPN1 and blocks development of P. falciparum via a new epitope.
Figure 7: Location of predicted B- and T-cell epitopes on domain I of AnAPN1.

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Acknowledgements

We thank the staff at the MX2 beamline of the Australian Synchrotron for assistance with X-ray data collection and the Monash Macromolecular Crystallization Facility for initial crystallization experiments. We also thank Y. Mok at the Macromolecular Interactions Facility at the University of Melbourne for assistance with sedimentation velocity experiments and J. Plieskatt at George Washington University for valuable comments and suggestions. We thank P. Eggleston and H. Hurd (both at Keele University) for the A. gambiae KEELE strain. Last, but not least, we thank the children, parents and the community of Mfou for their eager participation in this study. The work was funded in part by the PATH-Malaria Vaccine Initiative and Bloomberg Family Foundation through the Johns Hopkins Malaria Research Institute (to R.R.D.). J.S.A. was supported as a Johns Hopkins Malaria Research Institute predoctoral fellow, and D.K.M. was supported as a Calvin and Helen Lang Postdoctoral Fellow in the Biological Sciences. M.M.S. is supported by an Institut de Recherche pour le Développement Fellowship. B.B.T. is funded by the Ifakara Health Institute. S.C.A. is supported by an Australian National Health and Medical Research Council Early Career Fellowship (1072267). N.A.B. is supported by an Australian Research Council Future Fellowship (110100223). This publication was also made possible by the US National Institutes of Health National Center for Research Resources (UL1 RR 025005).

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Author notes

  1. Sarah C Atkinson and Jennifer S Armistead: These authors contributed equally to this work.

  2. Rhoel R Dinglasan and Natalie A Borg: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Sarah C Atkinson & Natalie A Borg

  2. W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

    Jennifer S Armistead, Derrick K Mathias, Dingyin Tao, Nahid Borhani-Dizaji, Brian B Tarimo & Rhoel R Dinglasan

  3. Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

    Jennifer S Armistead, Derrick K Mathias, Dingyin Tao, Nahid Borhani-Dizaji, Brian B Tarimo & Rhoel R Dinglasan

  4. Laboratoire de Recherche sur le Paludisme, Institut de Recherche pour le Développement–Organisation de Coordination et de Coopération pour la Lutte Contre les Grandes Endémies en Afrique Centrale, Yaoundé, Cameroon

    Maurice M Sandeu & Isabelle Morlais

  5. Nelson Mandela African Institute for Science and Technology, Arusha, Tanzania

    Brian B Tarimo

  6. Ifakara Health Institute, Dar es Salaam, Tanzania

    Brian B Tarimo

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  1. Sarah C Atkinson
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Contributions

S.C.A., J.S.A., D.K.M., M.M.S., D.T., N.B.-D. and I.M. designed and performed experiments. S.C.A., J.S.A., R.R.D. and N.A.B. analyzed structural and biochemical data. B.B.T., I.M. and R.R.D. analyzed all functional data. S.C.A., J.S.A., R.R.D. and N.A.B. led the manuscript preparation, with critical contributions from all authors.

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Correspondence to Rhoel R Dinglasan or Natalie A Borg.

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Integrated supplementary information

Supplementary Figure 1 Zinc and peptide within the AnAPN1 active site.

(a) Omit map for peptide in active site of molecule A, contoured at 1.8 σ. (b) Omit map for peptide in active site of molecule B, contoured at 1.6 σ. (c) Active site of AnAPN1 (molecule A) with zinc specific DANO map shown in pink, contoured at 7 σ. Key catalytic residues His366, His370 (both blue) and Glu389 (orange), as well as the active site bound peptide (green) are shown. Zinc is shown as a gray sphere.

Supplementary Figure 2 Sequence alignment of AnAPN1 and porcine APN showing the location of secondary-structure elements of AnAPN1.

Blue shading indicates positions that have fully conserved residues. The β-strands are shown as arrows and α-helices as blocks. AnAPN1 and porcine APN have moderate sequence identity (32% overall). Sequences were aligned with Clustal Omega and the alignment was edited and annotated using Jalview (version 2.8.1) and Adobe Illustrator CC.

Supplementary Figure 3 Different arrangements of domain IV from various aminopeptidases.

Surface representation of (a) AnAPN1, with a large cleft between domains II and IV, which is similar to that observed for (b) porcine APN (PDB ID 4FKE11), and (c) the open conformation of ERAP1 (pale blue, PDB ID 3MDJ14), but distinct from (d) human APN (yellow, PDB ID 4FYQ17) and (e) closed ERAP1 (bright blue, PDB ID 2YD0 (ref. 13)).

Supplementary Figure 4 Sedimentation velocity analyses of AnAPN1.

(a) Absorbance at 230 nm plotted as a function of radial position (cm) for AnAPN1 at an initial concentration of 0.15 mg/mL. Raw data (open circles) are plotted at time intervals of 8 mins and are overlaid with the continuous size-distribution best-fit (solid line) shows in (c). (b) Continuous sedimentation coefficient distribution (c(s)) plotted as a function of standardized sedimentation coefficient (s20,w) for AnAPN1 at 0.15 mg/mL. (c) Continuous mass distribution (c(M)) plotted as a function of molar mass (kDa) for AnAPN1 at 0.15 mg/mL.

Supplementary Figure 5 Epitope and functional activity analyses of monoclonal antibodies against the E. coli–expressed recombinant NT135aaAnAPN1.

(a-b) Peptide ELISA analyses of 2A12 and 4H5B7 epitope specificity. Gray bars are normal mouse IgG controls and black bars represent respective mAbs. (c) Replicate studies demonstrating the lack of transmission-blocking activity for mAbs 2A12 and 2C6 as compared to normal mouse (NM) serum IgG as measured by the Standard Membrane Feeding Assay (SMFA). (d) Immunoblot analysis of 4H5B7 mAb recognition of recombinant NT135aaAnAPN1 (double arrowhead) and near full-length AnAPN1 (single arrowhead), which was used for structural characterization. (e) The transmission-blocking activity of peptide 5-specific IgG (Pep 5 only) from NHPs that were immunized with the E. coli-expressed recombinant NT135aaAnAPN1. Peptide 5-specific IgG was depleted (P5-dep) from pooled NHP serum. (f) The transmission-blocking activity of NHP anti-Pep 7 IgG isolated by affinity chromatography (10 µg/mL) and 500 μg/mL of total IgG from mice following immunization with KLH-P7. (g-h) The transmission-blocking activity of serial dilutions of mAb 4H5B7 as compared to control mAb 2A12 (25 µg/mL) as measured by SMFA in An. stephensi NIH strain (g) and An. stephensi Nijmegen strain (h). The horizontal bars in panels c, e, g, h represent median oocyst number per mosquito midgut for each group and panel f is mean oocyst intensity. All SMFA data were analyzed statistically using a zero-inflated Generalized Linear Mixed Model as appropriate and the corresponding P-values and raw data are found in Source Data.

Source data

Supplementary Figure 6 Clustal Omega sequence alignment of APN from Bombyx mori versus AnAPN1.

Blue shading indicates positions that have fully conserved residues. The Cry1Aa toxin binding site on B. mori (NP_001037013.1) is denoted by a cyan bar above the sequences, while purple and green bars denote AnAPN1 peptides 7 and 9, respectively.

Supplementary Figure 7 Purification scheme for near-full-length recombinant AnAPN1.

(a) Chromatogram of HPLC-SEC separation of crude recombinant AnAPN1 secreted from S2 cells. (b) SDS-PAGE of HPLC fractions visualized on a Li-Cor Odyssey scanner. Peak 1 = HPLC peak at time 10.73 min (contaminating protein) and Peak 2 = HPLC peak at time 12.37 min (recombinant AnAPN1).

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Atkinson, S., Armistead, J., Mathias, D. et al. The Anopheles-midgut APN1 structure reveals a new malaria transmission–blocking vaccine epitope. Nat Struct Mol Biol 22, 532–539 (2015). https://doi.org/10.1038/nsmb.3048

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