RSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine

doi:10.1016/j.nano.2016.08.006

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 13, Issue 2, February 2017, Pages 411-420

https://doi.org/10.1016/j.nano.2016.08.006 Get rights and content

Abstract

Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infections in children, yet no vaccine is available. The sole licensed preventive treatment against RSV is composed of a monoclonal neutralizing antibody (palivizumab), which targets a conformational epitope located on the fusion protein (F). Palivizumab reduces the burden of bronchiolitis but does not prevent infection. Thus, the development of RSV vaccines remains a priority. We previously evaluated nanorings formed by RSV nucleoprotein (N) as an RSV vaccine, as well as an immunostimulatory carrier for heterologous antigens. Here, we linked the palivizumab-targeted epitope (called FsII) to N, to generate N-FsII-nanorings. Intranasal N-FsII immunization elicited anti-F antibodies in mice that were non-neutralizing in vitro. Nevertheless, RSV-challenged animals were better protected against virus replication than mice immunized with N-nanorings, especially in the upper airways. In conclusion, an N-FsII–focused vaccine is an attractive candidate combining N-specific cellular immunity and F-specific antibodies for protection.

Graphical Abstract

Here, we describe the development of a nanoparticle vaccine against RSV, based on a neutralizing epitope located on the antigenic site II of RSV fusion glycoprotein (highlighted in red on left panel). This epitope was fused to a vaccinal carrier composed of nanometric rings formed by RSV nucleoprotein (middle panel). The protective potency of this nanoparticle vaccine was evaluated in vaccinated mice using a recombinant RSV that express luciferase as a challenge virus (right panel).

Section snippets

Plasmid construction, protein expression and purification

N-FsII (Nter) and N-FsII (G106) fusion proteins were engineered by inserting the FsII epitope sequence (STYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQS) either at the N-terminus or between residues G106 and K107 of N protein, respectively. For N-FsII (Nter), the cDNA coding for FsII epitope (Long strain) was amplified by PCR using DreamTaq™ polymerase (Thermo Scientific) and cloned at NheI and BamH1 restriction sites in pET-N plasmid.²⁷ For N-FsII (G106), a KpnI restriction site was created by

Design and characterization of F epitope-fused N-nanorings (N-FsII)

FsII sequence (amino-acids S248 to S285) was fused to RSV N. Two anchoring sites were investigated: the N-terminus (Nter) and an exposed loop (position G106-K107) (Figure 1, A). The insertion between G106 and K107, which form the loop at the top of the β-hairpin projecting away from the N-terminal domain of N, was designed to stabilize FsII and to maintain its antigenic conformation. The two N-FsII proteins [(Nter) and (G106)] were analyzed by SDS-PAGE. A single band migrating at the expected

Discussion

The main objective of this work was to evaluate the potency of nanoparticles generated by RSV N (N-nanorings) as a vaccinal carrier to present a conformational neutralizing epitope of RSV F.

Our first goal was to display an epitope of the human RSV F antigenic site II (FsII, target of palivizumab) on N-nanorings, in a way to maintain its antigenic tertiary structure. In a previous attempt to design chimeric N-nanorings fused to bovine FsII, we failed to generate anti-F antibodies in calves,

Acknowledgments

We thank Jérôme Pottier, Mathilde Beauducel, Marlène Héry and Charline Pontlevoy from the Fish and Rodent Experimental Infectiology unit (IERP, INRA, Jouy-en-Josas, France), for their support in the animal facilities. We also thank Marie Galloux and Christophe Chevalier for their important support and for providing pET-N and pGEX-PCT plasmids. We thank the MIMA2 platform for access to the IVIS-200, which was financed by the Ile de France region (SESAME). We thank Geraldine Taylor from the

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Most probably this protection was mediated by a cellular response [28]. Indeed, although high levels of N-specific antibodies were induced, they were not neutralizing and their role in protection was not demonstrated [29]. It is to be expected that a RSV vaccine based on the sole induction of a cellular response, and therefore permissive for a first cycle of infection, is unlikely to be accepted by medical authorities and would not be relevant for a booster approach, especially for in utero transfer by pregnant mothers.
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: Funding: Agence Nationale de la Recherche (ANR-12-RPIB-0004-02).

: Conflicts of interest: The authors declare that there are no conflicts of interest.

¹: Present address: DBV Technologies, 177–181, avenue Pierre Brossolette, 92,120, Montrouge, France.

²: Present address: GenoSafe, 1 bis rue de l'International, 91,000, Evry, France.

³: Present address: Versailles Saint-Quentin-en-Yvelines University, 78,280, Guyancourt, France.

⁴: J.F.E. and S.R. shared last co-authorship.

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Original ArticleRSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine

Abstract

Graphical Abstract

Section snippets

Plasmid construction, protein expression and purification

Design and characterization of F epitope-fused N-nanorings (N-FsII)

Discussion

Acknowledgments

Virus Res

Curr Opin Virol

J Mol Biol

Vaccine

Vaccine

Mol Immunol

Vaccine

Procedia Vaccinol

Mol Ther - Methods Clin Dev

The pathogenesis of respiratory syncytial virus disease in childhood

Br Med Bull

Lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease

Nat Med

Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function

J Virol

Safety and effectiveness of palivizumab in children at high risk of serious disease due to respiratory syncytial virus infection: a systematic review

Infect Dis Ther

Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants

Pediatrics

Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies

J Gen Virol

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial

J Gen Virol

Neutralization of human respiratory syncytial virus infectivity by antibodies and low-molecular-weight compounds targeted against the fusion glycoprotein

J Virol

Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Proc Natl Acad Sci U S A

Original Article
RSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine