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A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein

Nature Microbiology volume 2, Article number: 16271 (2017) Cite this article

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Abstract

Respiratory syncytial virus (RSV) remains a major human pathogen, infecting the majority of infants before age two and causing re-infection throughout life. Despite decades of RSV research, there is no licensed RSV vaccine. Most candidate vaccines studied to date have incorporated the RSV fusion (F) surface glycoprotein, because the sequence of F is highly conserved among strains of RSV. To better define the human B cell response to RSV F, we isolated from a single donor 13 new neutralizing human monoclonal antibodies (mAbs) that recognize the RSV F protein in the pre-fusion conformation. Epitope binning studies showed that the majority of neutralizing mAbs targeted a new antigenic site on the globular head domain of F, designated here antigenic site VIII, which occupies an intermediate position between the previously defined major antigenic sites II and site Ø. Antibodies to site VIII competed for binding with antibodies to both of those adjacent neutralizing sites. The new mAbs exhibited unusual breadth for pre-fusion F-specific antibodies, cross-reacting with F proteins from both RSV subgroups A and B viruses. We solved the X-ray crystal structure of one site VIII mAb, hRSV90, in complex with pre-fusion RSV F protein. The structure revealed a large footprint of interaction for hRSV90 on RSV F, in which the heavy chain and light chain both have specific interactions mediating binding to site VIII, the heavy chain overlaps with site Ø, and the light chain interacts partially with site II.

RSV expresses three surface proteins: attachment (G), small hydrophobic (SH) and fusion (F) proteins. The G and F glycoproteins are the targets of neutralizing antibodies. Although the RSV G protein does induce neutralizing antibodies, antigenic diversity in G proteins among RSV strains makes it difficult to design a broadly protective vaccine candidate based on immunogenicity to this protein. Although there is no licensed RSV vaccine, a prophylactic monoclonal antibody (mAb), palivizumab1 (Synagis; MedImmune), is available for prophylactic treatment of high-risk infants, yet the high cost and moderate efficacy limit its use. The F protein is a class I fusion glycoprotein that adopts two conformations during viral infection. The pre-fusion F conformation is metastable and is triggered easily to the post-fusion conformation, resulting in a dramatic change involving the formation of a six-helix bundle extending the hydrophobic fusion peptide into the host cell membrane2. Recent structural breakthroughs in X-ray crystallography have provided atomic-resolution detail of the post-fusion and pre-fusion F conformations3,4. Furthermore, structure-based design of the F protein has resulted in stabilized F constructs (Ds-Cav1 and SC-TM) that retain components of the pre-fusion F conformation and induce neutralizing antibody immune responses5,6. Several neutralizing antigenic sites have been reported previously, recognized by the representative mAbs 131–2a (ref. 7) (site I), palivizumab1 and motavizumab8 (site II), 101F (ref. 9) (site IV), 7.936 (ref. 10) (site V, near amino acid 447), 7.916 and 9.432 (ref. 10) (site VI, near amino acid 432) and the recently discovered pre-fusion specific mAb D25 (ref. 4) (site Ø). Furthermore, a quaternary-dependent pre-fusion-specific epitope has been described using mAb AM14 (ref. 11), as well as an RSV/metapneumovirus cross-neutralizing region near site II (ref. 12). Antigenic sites II and IV–VI are retained in both the pre- and post-fusion conformations of F (ref. 13), as evidenced by the X-ray structures having exposed epitopes at these sites in both conformations. Antigenic site Ø is pre-fusion-specific, as the conformational epitope is lost in the rearrangement to the post-fusion conformation.

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Figure 1: Epitope binning for the RSV F-specific mAbs.
Figure 2: X-ray crystal structure of hRSV90-RSV F SC-TM complex.
Figure 3: Comparison between hRSV90 and known antigenic sites and interactions between hRSV90 and pre-fusion RSV F.

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Acknowledgements

The authors thank members of the Crowe Laboratory, especially R. Bombardi, N. Murphy and G. Sapparapu, for technical assistance. A portion of the experiments described here used the Vanderbilt robotic crystallization facility, which was supported by NIH grant S10 RR026915. J.J.M. was supported by T32 AI 07474, NIAID and National Institutes of Health (NIH). The described project was supported by CTSA award no. UL1TR000445 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the NIH.

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Authors and Affiliations

  1. Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, 37232, Tennessee, USA

    Jarrod J. Mousa, Nurgun Kose, Pranathi Matta, Pavlo Gilchuk & James E. Crowe Jr

  2. Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, 37232, Tennessee, USA

    James E. Crowe Jr

  3. Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, 37232, Tennessee, USA

    James E. Crowe Jr

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  1. Jarrod J. Mousa
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Contributions

J.J.M. and J.E.C. designed the experiments and interpreted the data. J.J.M. expressed, purified, crystallized, collected data and solved the crystal structure of the hRSV-90-RSV F SC-TM complex. J.J.M. and N.K. generated hybridomas by electrofusion. P.M. expanded hybridoma cell cultures. J.J.M. and P.M. performed neutralization experiments and J.J.M. performed competition-binding and ELISA binding studies. P.G. performed self-reactivity assays. J.J.M. and J.E.C. wrote the manuscript. All authors reviewed and edited the manuscript.

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Correspondence to James E. Crowe Jr.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9, Supplementary Table 1. (PDF 6535 kb)

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Mousa, J., Kose, N., Matta, P. et al. A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein. Nat Microbiol 2, 16271 (2017). https://doi.org/10.1038/nmicrobiol.2016.271

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