Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

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Abstract

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix–loop–helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.

Introduction

Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants, resulting in more than 2 million children under the age of 5 years requiring medical attention each year in the United States.1 Worldwide, RSV is estimated to cause more than 30 million lower respiratory tract infections and more than 60,000 deaths annually.2 An effective vaccine has eluded researchers since the 1960s, when a candidate vaccine composed of formalin-inactivated virus increased disease severity in infants upon natural infection with RSV.3 Animal models suggest vaccine-enhanced disease and pathology was associated with an imbalanced TH2 response4, 5 and elicitation of low-affinity antibodies.6 Passive immunization studies and subsequent clinical use of the monoclonal antibody palivizumab (Synagis®; Medimmune, Gaithersburg, MD) have demonstrated that neutralizing antibodies against a single antigenic site on the fusion (F) glycoprotein can prevent severe disease caused by RSV.7 Thus, an effective RSV vaccine should elicit potent neutralizing antibodies while avoiding an imbalanced T-cell response. Current vaccines have been designed to promote CD8+ and TH1 T-cell responses in addition to neutralizing antibody responses. However, a vaccine that could mimic passive antibodies and could induce neutralizing antibodies without RSV-specific T-cell responses would be an attractive option, particularly for the neonate.

We hypothesized that immunogens designed to elicit antibodies that target specific neutralizing epitopes would fulfill these vaccine requirements. RSV-neutralizing antibodies target two glycoproteins: G (attachment) and F (fusion) glycoproteins.8 Palivizumab targets a major antigenic site on the F glycoprotein,9 referred to as antigenic site II or site A, which encompasses residues 255–275.9, 10 Peptides corresponding to this region bind to palivizumab-like antibodies but fail to elicit neutralizing antibodies when injected in mice,11 suggesting that the free peptide fails to mimic the conformation of the epitope. In aqueous solution, the free peptide adopts a random-coil conformation but transitions to a helix–loop–helix conformation in the presence of 30% trifluoroethanol.12 It is this helical conformation that is recognized by neutralizing antibodies, as evidenced in the crystal structure of a potent palivizumab derivative, motavizumab, in complex with the peptide.13

The elicitation of structure-specific antibodies has recently been achieved by stabilizing the neutralizing-antibody-bound conformation of an epitope by using heterologous proteins as scaffolds to support the three-dimensional epitope structure.14, 15 These “epitope-scaffolds” mimicked the human immunodeficiency virus-1 gp41 epitopes of the broadly neutralizing antibodies 2F5 and 4E10 and, when used as immunogens, elicited polyclonal serum responses that recognized the structure of the epitope similarly to 2F5 and 4E10. Here we apply and extend this methodology to the motavizumab epitope, which can be considered a complex epitope consisting of residues from two separate α-helices. A new computational method was derived for identifying scaffold proteins that are capable of supporting such a discontinuous epitope structure. Three motavizumab epitope-scaffolds were designed, and their biochemical, biophysical, and immunological properties were characterized. The results have implications for the structure of the motavizumab epitope on the F glycoprotein, RSV vaccine development, and antibody-mediated RSV neutralization.

Section snippets

Computational motavizumab epitope transplantation

Analysis of the previously determined crystal structure of motavizumab bound to its RSV F peptide epitope13 identified 13 discontinuous RSV F residues that were contacted by motavizumab (Fig. 1a). We hypothesized that these 13 residues would be sufficient for eliciting motavizumab-like antibodies provided that their three-dimensional arrangement was preserved. Since all 13 residues were located on two α-helices (and none on the connecting loop), the motavizumab epitope could be considered a

Discussion

The difficulty in creating an RSV vaccine was increased by the demonstration of vaccine-enhanced illness in infants immunized with formalin-inactivated RSV,3 which was due in part to the elicitation of a deleterious T-cell response5 and low-affinity antibodies.6 Newer vaccine candidates using either full-length sequences or peptides as antigens and presented as a purified polypeptide, a gene-based vector, a virus-like particle, or an attenuated virus will elicit both neutralizing antibodies and

Multisegment side-chain grafting

We previously devised a computational method for the transplantation of continuous epitopes, called ‘side-chain grafting.14, 15 To allow identification of ‘side-chain grafting’ scaffolds for the discontinuous motavizumab epitope, we extended the Rosetta-implemented matching stage14 to allow searches for backbone superposition over multiple discontinuous segments. This method, called “multisegment side-chain grafting,” conducts the matching stage in a similar manner as the original

Acknowledgements

The authors would like to thank the staff at SER-CAT for help with X-ray diffraction data collection and the members of the Structural Biology Section and Structural Bioinformatics Section at the Vaccine Research Center for insightful comments and discussions. Support for this work was provided by the Intramural Research Program of the National Institutes of Health (National Institute of Allergy and Infectious Diseases). B.E.C. was supported by a fellowship from the Portuguese Fundação para a

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    J.S.M., B.E.C., and M.C. contributed equally to this work.

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