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. 2012;7(3):e34331.
doi: 10.1371/journal.pone.0034331. Epub 2012 Mar 29.

The respiratory syncytial virus G protein conserved domain induces a persistent and protective antibody response in rodents

Thien N Nguyen  1 , Ultan F Power, Alain Robert, Jean-François Haeuw, Katia Helffer, Amadeo Perez, Miguel-Angel Asin, Nathalie Corvaia, Christine Libon
Affiliations

The respiratory syncytial virus G protein conserved domain induces a persistent and protective antibody response in rodents

Thien N Nguyen et al. PLoS One. 2012.

Abstract

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130-230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Central conserved domain of RSV G attachment protein (aa130–230).
We present here the aa sequence of the conserved domain of (A) G2Na in a hypothetical schematic 2D structure with the highly conserved 13 aa domain followed by the Cysteine noose (dotted circle) [sequence in brackets with Cys res. in bold]. Note the disulphide bridge between the Cys res. and the Lys rich heparin binding domain. (B) G2Na derived from RSV-A (Long) G protein compared to G2Nb derived from RSV-B (8/60) G protein from aa121–240. Conserved domains are underlined. Cys res. positions in bold letters are respectively 173, 176, 182 and 186. (* divergent).
Figure 2
Figure 2. Schematic presentation of recombinant G protein derivatives.
The gene encoding G2Na was fused to (A) DT derivatives resulting in G2NaDTa, G2NaDTb and G2NaDTaDTb. DTa was subcloned the nucleotide sequence encoding (aa1–185) of the catalytic domain of DT as described in Materials and Methods. Note that Gly res. at aa52 was substituted by Glu in order to inactivate DT toxicity. DTb was subcloned from the nucleotide sequence encoding (aa202–456) of the transmembrane domain of DT. DTaDTb is a fusion product of DTa to DTb. (B) G2b (aa130–230) of RSV-B G protein (8/60 strain) resulting in G2ab: in this fusion form, G2Na has 4 conserved Cys but G2b has only 2 conserved Cys176 and 182, the 2 outer Cys173 and Cys186 were substituted by Ser res. in order to avoid spurious disulphide bridges. G2Nb used for admixing with G2Na has all four Cys res.
Figure 3
Figure 3. Immunogenicity and protective efficacy against RSV-A conferred by G2Na, BBG2Na, G2NaDTa, G2NaDTb and G2NaDTaDTb in cotton rats.
Groups of animals were immunised thrice im with G2Na derivatives according to the outlined experimental protocol (A). After the 3rd injection, on D60, we determined by ELISA the (B) anti-G2Na IgG Ab titre, (C) anti-RSV-A (Long) IgG Ab titre. Five days after challenge with RSV-A (105 TCID50) we evaluated protective efficacy by determining the RSV-A titre in the lungs of immunised animals. Data were expressed as Log10 TCID50/g lung tissue. The dotted line represents the detection limit of RSV-A in the lungs.
Figure 4
Figure 4. Immunogenicity of G2Na, G2Nb, G2ab and G2Na+G2Nb in mice.
Groups of animals were immunised im twice with Ag/Alum according to experimental protocol outlined in (A). We determined ELISA IgG Ab titre induced respectively by G2Na (dark blue), G2Nb (white), G2ab (stripe) and G2Na+G2Nb (light blue) on D29, D45 and D60, against (B) G2Na (C) G2Nb and on D60 (D) RSV-A (Long) on the left panel and RSV-B (8/60) on the right panel.
Figure 5
Figure 5. Immunogenicity and protective efficacy conferred by G2Na against heterologous challenge in mice.
Groups of mice were immunised ip twice with either 0.5 µg or 3 µg of Ag in Alum according to the experimental protocol outlined in (A). After the 2nd injection, we determined (B) by ELISA IgG Ab titre against G2Na (light blue) left panel, RSV-A (dark blue) middle panel and RSV-B (green) right panel. Immunised mice were divided into 2 groups (n = 5), before challenge with either RSV-A (105 TCID50) and RSV-B (106 TCID50). Protective efficacy was evaluated (C) by determining the titre in the lungs (expressed as Log10 TCID50/g lungs) of RSV-A (left panel, in dark blue) and RSV-B (right panel, in green). Dotted line represents the limited detection of RSV in the lungs.
Figure 6
Figure 6. Persistence of Ab induced by G2Na in cotton rats and protective efficacy against RSV-A.
Groups of animals were immunised im twice by Ag/Alum according to the experimental protocol outlined in (A). We studied the duration of Ab induced (B) by determining IgG Ab titre up to D148 against RSV-A Long strain (blue circle), RSV-A BT2a clinical strain (red square) and RSV-B CH18537 strain (green triangle). Protective efficacy of G2Na Ab induced against RSV-A challenge (105TCID50) was evaluated in comparison to Synagis® (C) by determining titre (expressed as Log10 (TCID50)/g lungs titre of immunised and treated animals. From left to right: PBS control immunised group, G2Na immunised group and Synagis® (5 mg/Kg) treated group.
Figure 7
Figure 7. Persistence and affinity maturation of Ab induced by G2Na/AL and G2Na/PLGA in RSV-A primed mice.
Groups of primed-mice were immunised im once according to the experimental protocol outlined in (A). We monitored Ab induction on D0, D7, D28, D56, D92 and D148 by ELISA IgG (B) anti-RSV-A IgG induced by empty PLGA (white bars), by G2Na/AL (blue bars), and by G2Na/PLGA (striped blue bars). Results are expressed as Arbitrary Units (AU)/ml using a positive control serum as a reference. * p<0.05. We determined affinity maturation of each serum Ab (see Materials and Methods) and summarised it in (C) bar graphic representation of % of animals with low avidity Ab (<3 M) in yellow and with high avidity Ab (≥3 M) in red on D28 (left panel) and on D148 (right panel). In the panels from left to right: empty PLGA group, G2Na/AL group and G2Na/PLGA group.
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