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. 2012 Feb 21;109(8):3089-94.
doi: 10.1073/pnas.1115941109. Epub 2012 Feb 8.

Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention

Margarita Magro  1 , Vicente Mas, Keith Chappell, Mónica Vázquez, Olga Cano, Daniel Luque, María C Terrón, José A Melero, Concepción Palomo
Affiliations

Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention

Margarita Magro et al. Proc Natl Acad Sci U S A. .

Abstract

Human respiratory syncytial virus (hRSV) is the most important viral agent of pediatric respiratory infections worldwide. The only specific treatment available today is a humanized monoclonal antibody (Palivizumab) directed against the F glycoprotein, administered prophylactically to children at very high risk of severe hRSV infections. Palivizumab, as most anti-F antibodies so far described, recognizes an epitope that is shared by the two conformations in which hRSV_F can fold, the metastable prefusion form and the highly stable postfusion conformation. We now describe a unique class of antibodies specific for the prefusion form of this protein that account for most of the neutralizing activity of either a rabbit serum raised against a vaccinia virus recombinant expressing hRSV_F or a human Ig preparation (Respigam), which was used for prophylaxis before Palivizumab. These antibodies therefore offer unique possibilities for immune intervention against hRSV, and their production should be assessed in trials of hRSV vaccines.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Induction of binding and neutralizing antibodies in rabbits immunized with recombinant vaccinia viruses expressing different forms of hRSV_F. Serial dilutions of sera from rabbits inoculated with either Vac/Fc (α-Fc) or Vac/FTM- (α-FTM-) were tested for binding to purified FTM- adsorbed to ELISA plates (A) or hRSV neutralization (B). Control serum from a preimmune rabbit was included as control. Total α-Fc antibodies were purified by protein A-Sepharose chromatography from the sera of rabbits immunized with Vac/Fc. The α-Fc antibodies were loaded onto a column of FTM-Sepharose, and the unbound antibodies (α-Fc/ΔFTM-) were collected and saved. After washing, the antibodies bound to the column (α-Fc/FTM-) were eluted. The α-Fc, α-Fc/ΔFTM-, and α-Fc/FTM- antibodies were then tested for binding to FTM- (C) and for hRSV neutralization (D). Total α-FTM- antibodies from rabbits inoculated with Vac/FTM- were processed similarly to yield the α-FTM-/ΔFTM- antibodies that did not bind to the FTM-Sepharose column and the α-FTM-/FTM- antibodies eluted from this column. α-FTM-, α-FTM-/ΔFTM-, and α-FTM-/FTM- antibodies were tested for binding to FTM- (E) and hRSV neutralization (F). Results are presented as means ± SEM of three independent experiments.
Fig. 2.
Fig. 2.
Rabbit α-Fc antibodies recognize HRSV_F expressed at the cell surface even if depleted of antibodies able to bind to FTM- (α-Fc/ΔFTM-). HEp2 cells were infected with hRSV (Long strain, multiplicity of infection 5 pfu per cell) and tested 48 h later by flow cytometry for cell surface labeling with α-Fc, α-Fc/ΔFTM-, and α-Fc/FTM- antibodies (A). Alternatively, HEp-2 cells were infected with recombinant vaccinia virus, either hRSV_P (Vac/P) or hRSV_Fc (Vac/Fc), and tested 24 h later by flow cytometry for cell surface labeling with α-Fc, α-Fc/FTM-, and α-Fc/ΔFTM- antibodies before (B) or after adsorption of the antibodies to cells infected with Vac/P (C) or Vac/Fc (D). Mock infected cells (dashed lines) were included always as control. Note the reactivity of α-Fc/FTM- antibodies with cells infected with Vac/P (B Right), although lower than the reactivity of α-Fc and α-Fc/ΔFTM- probably because anti-vaccinia antibodies that bound unspecifically to the FTM- column. Results are representative of at least three independent experiments.
Fig. 3.
Fig. 3.
α-Fc, α-Fc/ΔFTM-, and α-Fc/FTM- antibodies lost their neutralizing activity after incubation with cells infected with Vac/Fc. The three antibody preparations of Fig. 2 were depleted of antibodies binding to cells infected with either Vac/P (ΔVac) or Vac/Fc (ΔFc) and then tested either before or after the different adsorptions for binding to FTM- (A–C) and for hRSV neutralization (D–F). Although the antibodies incubated with cells infected with Vac/Fc were also depleted of anti-vaccinia antibodies (i.e., they were also ΔVac), they were labeled ΔFc to distinguish them of those incubated with cells infected with Vac/P (labeled ΔVac in this figure). Results are presented as means ± SEM of three independent experiments.
Fig. 4.
Fig. 4.
Stabilization of the prefusion form of hRSV_F with intermonomeric disulfide bonds. (A) Scheme of the hRSV_F primary structure, denoting the three main hydrophobic regions: signal peptide (SP), fusion peptide (FP), and transmembrane region (TM). Also indicated are the two furin cleavage sites (arrows) that generate the F2 and F1 chains and the cysteines (gray dots and numbers) that participate in the intramonomeric disulfide bonds observed in the pos-fusion structure of hRSV. HRA and HRB represent the heptad repeat sequences of the F1 chain. Partial sequences, shown below the scheme, highlight the asparagines (N, boldface) introduced to ablate furin cleavage in FcN and FcN2C-C constructs and the cysteines introduced in the latter construct (shown in orange) to make new intermonomeric disulfide bonds. The His-tag sequence added at the C terminus is shown above the scheme. (B) 3D model of the prefusion conformation of hRSV_F built with the coordinates of the PIV5_F protein (20) (PDB ID code 2B9B). Each monomer is colored differently. Cysteine residues (C) replacing residues 481, 489, 509, and 510 in FcN2C-C are represented by golden balls alternating in two of the monomers (magenta and blue). (C) Postfusion structure of hRSV_F, reported by MacLellan et al. (26) (PDB ID code 3RRR). Colors and locations of engineered cysteine residues are presented as in B. Purified FcN, FcN2C-C, and FTM- proteins (Methods) were resolved by SDS/PAGE and either stained with Coomassie blue (D) or electrotransferred to nylon membranes and stained by Western blot with α-FTM- antibodies (E). Electron microscopy of the three purified proteins (F–H) negatively stained with uranyl acetate. (Scale bar: 50 nm.) Two-times-magnified selected molecules are shown in Insets.
Fig. 5.
Fig. 5.
The stabilized prefusion hRSV_F protein is bound by rabbit and human neutralizing antibodies depleted of those that recognize FTM-. (A) The three proteins, FTM-, FcN, and FcN2C-C of Fig. 4 were bound to ELISA plates via a bridge made with MAb 101F and then tested for binding to the antibodies indicated at the bottom of each image. α-Fc, α-FTM-, and α-Fc/ΔFTM- have been described in Fig. 1. The α-6HB antibodies (Methods) were raised in rabbits inoculated with a bacterially expressed construct that mimics the six-helix bundle present in the postfusion form of hRSV_F (28). RG is the Ig preparation (Respigam) used some years ago in the clinic (a generous gift of MedImmune). A sample of this preparation was also depleted of antibodies binding to FTM-Sepharose (RG/ΔFTM-). (B) RG was depleted sequentially of antibodies binding to FTM-Sepharose (RG/ΔFTM-), G-Sepharose (RG/ΔFTM-/ΔG), and cells infected with either Vac/Fc (RG/ΔFTM-/ΔG/ΔFc) or Vac/FcN2C-C (RG/ΔFTM-/ΔG/ΔFcN2C-C), and all these antibody preparations were used in a microneutralization test with hRSV. C- is a preimmune rabbit antibody. (C) FTM-, FcN, and FcN2C-C proteins were adsorbed directly to microtiter plates and used in an ELISA with the four MAbs indicated in each image. 2F, 47F, and 101F are murine MAbs that recognize nonoverlapping epitopes in hRSV_F (29) Palivizumab (a generous gift of Abbott España) is a humanized MAb in use as a prophylactic treatment for hRSV infections. Escape mutants selected with Palivizumab partially overlap those selected with MAb 47F (30), and both antibodies bind to similar peptides of hRSV_F (31, 32).
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