Immunology Letters 57 (1997) 15–17

Peptide mimics of a conformationally constrained protective epitopesof respiratory syncytial virus fusion protein

D. Chargelegue a,*, O.E. Obeid a, D.M. Shaw 1,a, A.N. Denbury a, P. Hobby b, S.C. Hsu a,M.W. Steward a

a London School of Hygiene and Tropical Medicine, Molecular Immunology Unit, London WC1E 7HT, UKb Randall Institute, London, UK

Abstract

Aims: To identify peptides that mimic (mimotopes) conformational and protective epitopes of RSV fusion protein and to assesstheir efficacy as immunogens and potential vaccines. Material and methods: An 8-mer solid-phase (TG resin) library was screenedwith a neutralising and protective RSV fusion protein specific monoclonal antibodies (Mab-19). After selection of positive beads,reactive sequences were identified by microsequencing and 8-mer peptides were synthesised. Improvement of binding was analysedby amino acid replacement using the SPOTs method. Results: Mabs were not able to bind to the free and soluble peptides, nordid these peptides induce anti-RSV specific antibodies. However, several peptides re-synthesised on a TG resin (to producede-protected 8-mer peptides linked to the resin) or as SPOTs reacted specifically. Therefore it was critical to be able to reproducethis conformation in order to use these mimotopes as immunogens and potential vaccines. Using C-terminal constrained versionsof the mimotopes, strong binding of one of the Mabs to the peptides was demonstrated by surface-plasmon resonance.Immunisation of Balb/c mice with these peptide-mimics produced anti-sera that: (1) reacted specifically with RSV; (2) inhibitedthe binding of the Mab to the virus; (3) neutralised RSV in vitro with high titres (range: 80–640); and (4) reduce significantly theviral load in the lungs of mice challenged with RSV (PB0.01). Conclusions: This report demonstrates for the first time that: (1)a protective epitope of the conserved RSV fusion protein can be mimicked by synthetic peptides; and (2) immunisations with thesemimotopes induced specific anti-RSV neutralising antibodies and reduced viral load in vivo. These results represent a novelconcept for the development of a vaccine against RSV. Published by Elsevier Science B.V.

Keywords: RSV fusion protein; Immunogen; Vaccine

1. Overview

Respiratory syncytial virus (RSV) is the major causeof serious lower respiratory tract illness in infants andimmunosuppressed individuals world-wide and is esti-mated to be responsible for 65 million severe infectionsand more than one million deaths annually [1,2]. Theserious nature of this viral infection has prompted theWHO to designate it for rapid vaccine development [3].

Although the severity of disease declines with re-peated infection, previous infection with RSV does not

prevent illness following subsequent infections and it isapparent that immunity is incomplete. Furthermore,the use of a formalin-inactivated vaccine in children inthe 1960s led to potentiation of disease during a subse-quent epidemic. These observations highlight thedifficulties in developing a vaccine against RSV. How-ever, studies in experimental animals have shown thatRSV-specific neutralising antibodies can prevent infec-tion in the lungs when administered prophylactically [4]and the intravenous administration of pooled IgG,containing a high titre of neutralising antibodies, pre-vented the development of serious RSV lower respira-tory tract illness in high risk children [5,6]. Both the F(fusion) and G glycoproteins of RSV play a major rolein eliciting this humoral immunity. Active immuniza-

* Corresponding author. Tel.: +44 171 9272294/2293; fax: +44171 6374314; e-mail: D.Chargelegue@lshtm.ac.uk

1 Present address: TNO, Leiden, Netherlands.

0165-2478/97/$17.00 Published by Elsevier Science B.V. All rights reserved.PII S 0 1 5 -2478 (97 )00045 -X

D. Chargelegue et al. / Immunology Letters 57 (1997) 15–1716

tion with the conserved F glycoprotein induces pro-tective antibody responses [7,8] and passive immunisa-tion with neutralising anti-F monoclonal antibodiescan protect small animals from RSV infection [9,10].As a result of these studies, a neutralising and protec-tive monoclonal antibody (Mab-19) has beenreshaped and humanised and has been shown to bemore effective at protection than polyclonal anti-RSVantibodies in a rodent model [11]. More recently, thesafety and efficacy of the humanised Mab-19 hasbeen assessed in human volunteers [12]. These resultsindicate that the induction of antibodies to specificepitopes on the virus proteins may well be sufficientfor protection. We propose that the development of asynthetic peptide vaccine against RSV is a rationalalternative to the use of conventional vaccine strate-gies.

However, the identification of the epitopes recog-nised by the protective antibodies is critical for thedevelopment of a peptide-based vaccine. The fact thatprotective epitopes on RSV-F protein appear to beconformational in nature has made their identificationfrom the primary amino acid sequence virtually im-possible. Indeed, earlier attempts in our laboratoryand elsewhere using synthetic peptides representinglinear sequences of the F-protein (aa 205–225, 221–237, 261–273, 215–275, 417–438 and 481–491),failed to generate neutralising or protective humoralresponses [13–16]. Studies with peptides [17], antigenfragments [18] and antibody escape mutants [10]confirmed the involvement of both discontinuous anddistant residues within the F-protein, in epitopesrecognised by protective monoclonal antibodies [10].Thus, the use of alternative approaches for the iden-tification of these epitopes is essential.

Two types of random combinatorial peptide li-braries have been commonly employed to identifypeptide mimics (mimotopes) of conformational lig-ands: (1) a peptide library using filamentous phagefor the display of random sequences [19,20]; and (2)the resin-based ‘one bead one peptide’ library inwhich random peptides are synthesised on an insolu-ble solid-phase [21,22]. Phage peptide display librarieshave been used to identify core residues involved inlinear structural epitopes of HIV-1 gp120 (neutralis-ing), herpes virus D glycoprotein (protective) andHepatitis surface antigen [23–26] and we have re-cently used a solid-phase library to identify 8-merpeptides that mimic a protective epitope of measlesvirus F-protein [27]. The mimotopes identified by thisapproach do not bear any primary sequence relation-ship to the epitope on the virus but mimic its confor-mation and one induced a virus-neutralising andprotective antibody response [27]. Similarly, phagedisplayed peptide libraries have been used to identifysequences which mimic a discontinuous epitope of

hepatitis B surface antigen [28] and peptides that re-acted with Mabs specific for polysaccharides [29].

In our recent work, we have identified peptidemimics (mimotopes) of a conformational epitope ofRSV fusion protein recognised by Mab-19 using aresin based random peptide library of approximately14×106 peptide variants (D. Chargelegue et al.,manuscript in preparation). Mab-19 was not able tobind to the free and soluble peptides, nor did thesepeptides induce anti-RSV specific antibodies. How-ever, specific binding was demonstrated to depro-tected sequences uncleaved from the resin supportand to those synthesised on cellulose-paper in theSPOTs-ELISA format. Mimotopes with enhancedbinding to the Mab were selected by substitution ofamino acids in the original mimotope, followed byscreening using the SPOTs-ELISA and further vari-ants of the original sequences with enhanced bindingcharacteristics have been identified. To generate po-tential immunogens, the mimotopes were synthesisedas tetrameric Multiple Antigen Peptide (MAP) con-structs. Strong binding of the Mab-19 to these pep-tides was demonstrated with affinity constants in therange of 109 M−1 as estimated by surface-plasmonresonance. Preliminary studies have shown that im-munisation of mice with these constructs resulted inthe induction of anti-peptide antibodies which cross-reacted with RSV, inhibited Mab-19 binding to RSVand neutralised the virus in vitro (log2 titre: 6.32–9.32). Furthermore, systemic immunisation with oneof the constructs induced an antibody response whichresulted in a greater than 90% reduction in virus loadin lungs of BALB/c mice, following infection withRSV. To our knowledge this is the first evidenceshowing that peptide-mimics of a protective epitopeof the conserved RSV fusion protein can induce aspecific anti-RSV neutralising humoral response andreduce viral load in vivo. The results of these prelimi-nary studies certainly indicate the potential of thisapproach to the development of an RSV vaccine.Therefore, further studies are currently in progress inorder to determine the protective role of the antibodyresponse to these peptide mimics such as passive-transfer experiments [30] and polyclonal antibodyaffinity measurements [31,32]. Moreover we are inves-tigating the mucosal delivery of these mimotopesalone and together with a partially protective CTLepitope of the RSV M protein [33,34].

Acknowledgements

This work was supported by the LungFoundation and the UK Medical Research Council(ROPAs scheme).

D. Chargelegue et al. / Immunology Letters 57 (1997) 15–17 17

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