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Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies

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From Nature Microbiology

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Abstract

Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.

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Fig. 1: Strong binding of hPIV3 mAbs to surface glycoproteins of hPIV3.
Fig. 2: Potent neutralizing activity of PIV3 mAbs against PIV3 clinical isolates.
Fig. 3: HN-reactive mAb rPIV3-23 inhibits hPIV3 virus by diverse mechanisms, competition ELISA of HN-reactive mAbs and rPIV3-18 binding map to the pre-fusion F protein of hPIV3.
Fig. 4: Cryo-EM structure of the rPIV3-18 and hPIV3 F complex.
Fig. 5: Cryo-EM structure of the rPIV3-23 and rPIV3-28 Fabs bound to the hPIV3 HN dimer.
Fig. 6: PIV3 mAbs mediate prophylactic protection in cotton rats challenged with PIV3-CI-24 and synergize for neutralization.

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Data availability

All data needed to evaluate the conclusions in the paper are included in the paper or the Supplementary Information. Sequencing reads and consensus sequences have been deposited in NCBI under BioProject PRJNA992650. The atomic models used to determine and interpret the two protein structures detailed in this manuscript are available in the Protein Data Bank under the following accession codes: PDB 1V2I, PDB 4WEF, PDB 5I19, PDB 7BEL, PDB 3Q6G, PDB 5F9O, PBD 7DPM, PDB 6MJZ, PDB 7DPM. The cryo-EM structures and volume for the hPIV3 HN and hPIV3 F in complex with neutralizing Fabs have been deposited in the Protein Data Bank with the accession codes 8TQI (hPIV3 HN complex) and 8TQK (hPIV3 F complex). The electron density volumes are also available in the Electron Microscopy Data Bank under the codes EMD-41505 (hPIV3 HN complex) and EMD-41506 (hPIV3 F complex). Source data are provided with this paper.

Code availability

No new code was generated in this study. See the source data provided with this study and the deposited data.

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Acknowledgements

We thank members of the Jardetzky, Moscona and Crowe laboratories. The work was supported by NIAID/NIH grants R01 AI13752 and R01 AI114736. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy programme (U24 GM129541). The authors thank H. A. Khant of the S2C2 for invaluable support and assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Special acknowledgements to R. Irving for helping to procure cotton rats and helping with animal studies. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Naveenchandra Suryadevara, Ana Rita Otrelo-Cardoso.

  2. These authors jointly supervised this work: Theodore S. Jardetzky, James E. Crowe, Jr.

Authors and Affiliations

  1. Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN, USA

    Naveenchandra Suryadevara, Nurgun Kose, Elad Binshtein, Rachael M. Wolters, Laura S. Handal, Robert H. Carnahan & James E. Crowe Jr.

  2. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Ana Rita Otrelo-Cardoso, Yao-Xiong Hu & Theodore S. Jardetzky

  3. Department of Laboratory Medicine and Pathology, University of Washington Medical Center, Seattle, WA, USA

    Alexander L. Greninger

  4. Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA

    Robert H. Carnahan & James E. Crowe Jr.

  5. Departments of Pediatrics, Microbiology and Immunology, and Physiology and Cellular Biophysics, and Center for Host–Pathogen Interaction, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA

    Anne Moscona

  6. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    James E. Crowe Jr.

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  1. Naveenchandra Suryadevara
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Contributions

T.S.J. and J.E.C. conceived the project. N.S., A.R.O.-C., N.K., Y.-X.H., E.B., R.M.W., A.L.G. and L.S.H. performed laboratory experiments. A.L.G. and A.M. provided reagents. T.S.J., A.M. and J.E.C. obtained funding. R.H.C., T.S.J., A.M. and J.E.C. supervised the research. N.S., A.R.O., J.E.C. and T.S.J. wrote the first drafts of the paper. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Theodore S. Jardetzky or James E. Crowe Jr..

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Competing interests

J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, and BTG International Inc., is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols) and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda Vaccines, and IDBiologics during the conduct of the study. T.S.J. has served as a consultant for Pfizer. A.L.G reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen and Hologic, and research support from Gilead, outside of the described work. A.M. reports potential future financial interests in Thylacine Biotherapeutics Inc. unrelated to the present study. All other authors declare no competing interests. Vanderbilt University has applied for a patent for some of the antibodies in this paper (2024 International Patent Application No. PCT/US2024/028239 based on US Serial No. 63/504,549).

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Nature Microbiology thanks Michael Hoffmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Haemagglutination Inhibition assay and Fab neutralization.

1a. Haemagglutination Inhibition assay (Highlighted in Red- IgG, Blue -Fab, Green- RBCs only, Yellow - RBCs + PIV3 virus) 1b. Neutralization assay of PIV-3 virus using equimolar ratio IgG (30 µg/mL starting concentration) and Fabs (10 µg/mL starting concentration) of the PIV3 mAbs. Error bars indicate mean ± S.D.; data are representative of n = 2 independent experiments performed in technical triplicates.

Source data

Extended Data Fig. 2 Overview of the HPIV3 F and rPIV3-18 structure determination.

A. Comparison of the gel filtration profile for the HPIV3 F and the HPIV3 F: r-PIV3-18 complex. The complex was purified at least three times independently with consistent results. B. Motion corrected micrograph for HPIV3 F:rPIV3-18 complex (n = 11194). Two independent datasets were collected with similar results. C. Representative 2D class averages for HPIV3 F:rPIV3-18. D. FSC curves. E. Cryo-EM image processing flowchart. F. Local resolution map.

Extended Data Fig. 3 Overview of the HPIV3 HN and rPIV3-23:rPIV3-28 structure determination.

A. Comparison of the gel filtration profile for the HPIV3 HN and the HPIV3 HN: r-PIV3-23:rPIV3-28 complex. The complex was purified at least three times independently with consistent results. B. Motion-corrected micrograph for HPIV3 HN:rPIV3-23:rPIV3-28 complex (n = 7242). C. Representative 2D class averages for HPIV3 HN:rPIV3-23:rPIV3-28. D. FSC curves. E. Cryo-EM image processing flowchart. F. Local resolution map.

Extended Data Fig. 4 Overview of the HPIV3 HN and rPIV3-23 structure determination.

A. Gel filtration profile for the HPIV3 HN: r-PIV3-23 complex. The complex was purified at least two times independently with consistent results. B. Motion-corrected micrograph for HPIV3 HN:rPIV3-23 complex (n = 7524). C. Representative 2D class averages for HPIV3 HN:rPIV3-23. D. FSC curves. E. Comparison between the HPIV3 HN:rPIV3-23 density map (blue), the HPIV3 HN:rPIV3-23:rPIV3-28 atomic model (beige) and the HPIV3 HN:rPIV3-23:rPIV3-28 density map (gray).

Extended Data Fig. 5 Superimposition with HPIV3 HN dimer and tetramer structures.

A. Stereoview with superimposition of the HN complex with other paramyxovirus HN dimer structures (PDB IDs: 4WEF, 4MZA, 4MZE, 1V2I, 1V3B, 1V3D, 1V3C, 1V3E, 5KV9, 4XJR, 6C0M, 5KV8, 4XJQ and 1USR). B. Superimposition of the HPIV3 HN (light pink) +rPIV3-23 (purple) +rPIV3-28 (yellow) complex with the ‘4 heads down’ NDV HN tetramer (green) (PDB ID: 3T1E). C. Superimposition of the HPIV3 HN (light pink) +rPIV3-23 (purple) +rPIV3-28 (yellow) complex with the ‘2 heads up 2 heads down’ PIV5 HN tetramer (green) (PDB ID: 4JF7).

Extended Data Fig. 6 Comparison between the HPIV3 HN binding site with rPIV3-23 HCDR3 and the Neu5Ac.

A. Comparison between the HPIV3 HN binding site with rPIV3-23 (purple) HCDR3 and the Neu5Ac (PDB model ID 1V3C) bound. B. Comparison of Ulster strain NDV HN (old rose) with HPIV3 HN (light rose):rPIV3-23 (purple). Relevant residues are displayed as sticks. C. Superimposition of NDV HN dimer with Neu5Acen (yellow) and Neu5Ac (green) displayed as spheres (PDB ID 1USR). Glycan represented as sticks. A single rPIV3-28 Fab (yellow) is shown binding to the HN dimer for clarity.

Supplementary information

Reporting Summary

Supplementary Tables 1–3

Antibody variable gene usage and HCDR3 amino acid sequences. Inhibitory concentration50 (IC50, in ng ml−1) of PIV3 mAbs against clinical isolates. DRI value was estimated by CompuSyn.

Supplementary Table 4

Cryo-EM data collection and model refinement.

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. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

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Suryadevara, N., Otrelo-Cardoso, A.R., Kose, N. et al. Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies. Nat Microbiol 9, 2128–2143 (2024). https://doi.org/10.1038/s41564-024-01722-w

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