A foldon-free prefusion F trimer vaccine for respiratory syncytial virus to reduce off-target immune responses

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and older people. Current RSV subunit vaccines are based on a fusion protein that is stabilized in the prefusion conformation and linked to a heterologous foldon trimerization domain to obtain a prefusion F (preF) trimer. Here we show that current RSV vaccines induce undesirable anti-foldon antibodies in non-human primates, mice and humans. To overcome this, we designed a foldon-free RSV preF trimer by elucidating the structural basis of trimerization-induced preF destabilization through molecular dynamics simulations and by introducing amino acid substitutions that negate hotspots of charge repulsion. The highly stable prefusion conformation was validated using antigenic and cryo-electron microscopy analysis. The preF is immunogenic and protective in naive mouse models and boosts neutralizing antibody titres in RSV-pre-exposed mice and non-human primates, while achieving similar titres to approved RSV vaccines in mice. This stable preF design is a promising option as a foldon-independent candidate for a next-generation RSV vaccine immunogen.

Fig. 1. Foldon-containing preF protein induces off-target responses in mice, NHPs and humans.

a, RSV-pre-exposed NHPs (n = 10) were immunized at week 0, week 60 and week 189 (arrows) with preF protein or preF protein–Ad26-preF. Foldon-binding antibody titres were measured using ELISA in serum isolated before immunization and 4 weeks after every immunization. b, Human participants were immunized with preF protein combined with Ad26-preF at week 0 and with preF protein or preF protein–Ad26-preF (blue symbols, n = 10) or Ad26-preF alone (orange symbols, n = 6) at week 52 (arrows). Just before and 2 weeks after immunization, foldon-binding antibody titres were measured using ELISA. c, Balb/c mice were immunized twice with a 4 week interval with AREXVY, an AS01_E-adjuvanted preF-protein-based commercially available RSV vaccine (n = 6 per group) or buffer (n = 3). Foldon-binding antibody titres were measured using ELISA 2 weeks after the second immunization. End-point titres were calculated and expressed as log₁₀. The bars represent mean titres per group. The NHP and human ELISAs have no lower limit of detection (LLOD) defined while the mouse ELISA has an LLOD of 1.47 (log₁₀). NS, not statistically different. Statistical testing (two sided) was performed using paired t-test with Bonferroni correction (a), ANOVA with Bonferroni correction (b) or a Tobit model with Bonferroni correction (c). Source data

Fig. 2. Trimerization of RSV F destabilizes the prefusion conformation.

a, Multimerization profile of RSV-A F protein in the supernatant of Expi293F cells, as determined by native PAGE followed by western blot and probed with anti-RSV F antibody CR9506. RSV F proteins either were wild-type consensus sequences (‘WT’) or contained stabilizing substitutions D486N or E487Q. The presence or absence of a C-terminal foldon trimerization domain is indicated with a plus or a minus sign, respectively. The monomer and trimer bands are indicated. A stabilized (N67I, S215P, D486N) RSV preF trimer (‘preF’) with foldon was taken along as positive control for trimer formation. Native PAGE was performed three times with similar results. b, Analytical SEC analysis of RSV-A F protein expressed in the supernatant of Expi293F cells. RSV F proteins are as described in a, with the presence or absence of a C-terminal foldon trimerization domain indicated. Analytical SEC was performed 3 days after transfection at the day of collection (day 0) and after 2 and 7 days of storage at 4 °C (day 2 and day 7). As positive control for the preF trimer, a stabilized (N67I, S215P, D486N) construct was taken along with a foldon. The approximate retention time of trimeric (T) or monomeric (M) F is indicated. OD280, optical density at 280 nm. c, Antigenicity profile of wild-type, D486N or E487Q RSV-A F protein with (plus sign) or without (minus sign) a C-terminal foldon trimerization domain, as expressed in the supernatant of Expi293F cells. Measurements were performed with biolayer interferometry using a panel of monoclonal antibodies on the day of collection (day 0) and after 2 and 4 days of storage at 4 °C. The specificity of the anti-RSV F antibodies is indicated as binding to the preF or postF conformation, or as pan-specific (panF), indicating binding to both prefusion and postfusion conformations. R-equilibrium binding is reported as the average of n = 4 biological replicates + s.d., and individual data points are represented by open circles. Source data

Fig. 3. Regions of instability in RSV F form upon trimerization.

a, Left panels: RMSFs from MD simulations of wild-type RSV F monomer and trimer, projected on the PDB 4MMS trimer structure. The monomer fluctuations are projected on a trimer for easier comparison with the trimer simulation (central zoom on the fusion peptide region). Right panel: RMSF of the D486N mutant trimer. Also see Supplementary Fig. 3. The proteins are coloured according to the residue RMSF, with the colour white corresponding to the average fluctuations, blue to low fluctuations (less than mean − σ) and red to high fluctuations (more than mean + σ). b, Side view of the RSV F trimer. The architecture of the core is shown consisting of the negatively charged layer (red), aromatic layer (green) and positively charged layer (blue). c, Bottom view of the region of instability composed of the ring of six negative charges encircling the three-fold axis, depicting the concentrated area of electrostatic repulsion. D489 is shown as sticks. d, Positively charged region of instability at the interface of the protomers. Details of the interprotomeric repulsion between R106 and R339, the contribution of the F1 N-terminus positive charge and the positive charge at the edge of the aromatic ring of F137 are shown. Stabilizing interactions between Q354 and the F1 N-terminus are shown as dotted orange lines. Modelling of the wild-type RSV F trimer is as described in Methods.

Fig. 4. Stable prefusion RSV F without a foldon domain through stabilization of the fusion peptide cavity and HR2 region.

a, Analytical SEC analysis of RSV-A and RSV-B F protein with or without foldon, as determined in cell supernatant. RSV-A F carries substitutions N67I, S215P and D486N. RSV-B F carries substitutions P101Q, I152M, L203I, S215P, D486N and D489Y. Trimeric (T) or monomeric (M) F is indicated. b, PreF expression and stability depicted as trimer peaks in analytical SEC at day 0 versus the CR9501 binding ratio at day 0 and 6. Stabilizing substitutions were screened in a semi-stable consensus backbone (‘BB’; RSV-A F with foldon and D486N). Substitutions in blue were selected for follow-up. c, Analytical SEC on RSV-A and RSV-B F without a foldon (Δfoldon) in cell supernatant. RSV-A Δfoldon contains P101Q, S215P, Q354L, D486N, E487L and D489Y. RSV-B Δfoldon contains P101Q, I152M, L203I, S215P, Q354L, D486N, E487L and D489Y. T is indicated. d, Stability of indicated variants as determined by analytical SEC on supernatants after 15 min of incubation at indicated temperatures. The backbone is RSV-A Δfoldon described in c. e, Stability of indicated variants as determined by analytical SEC on supernatants stored for 0 or 16 days at 4 °C. The backbone is RSV-B Δfoldon described in c. f, SEC-MALS (~162 kDa) of RSV-A F Δfoldon trimers with stabilizing substitutions P101Q, S215P, Q354L, D486N, E487L, D489Y, F505W and S509F. Variants contain either a short HR2 (residues 1–513) or a long HR2 (residues 1–524). g, Melting temperature (Tm₅₀) of RSV-A F Δfoldon variants with a short or long HR2 sequence, as described in f, determined by differential scanning fluorimetry (DSF). Data reported as the average (blue line) of n = 3 technical replicates (grey lines). h, Antigenicity profile of RSV-A F Δfoldon variants with a short or long HR2, as described in f, measured by BLI. RSV postF protein is included as a control. Antibody specificity is indicated. The binding rate is reported as the average of three technical replicates, and individual data points are represented by open circles. Source data

Fig. 5. Cryo-EM analysis of stabilized RSV-A preF-Δfoldon.

a, Schematic overview of RSV F protein in green, fusion peptide (FP) in orange, HR1 in blue, loop 460–476 in salmon and HR2 in purple. The soluble F ectodomain used in experiments consists of residues 1–524 and lacks the transmembrane (TM) and cytoplasmic (CT) regions. b, Structure of the stabilized trimer, with two monomers shown as surface representation (grey) and one monomer as ribbons with colours according to a, and side chains of stabilizing substitutions shown as cyan spheres. Letters C–H refer to the view of panels c–h relative to b. c, Docking site shown in transparent surface of fusion peptide (orange) with yellow dotted lines showing the interactions of R339 with F137 and substitution 354L (cyan) stabilizing the turn in the fusion peptide. Grey spheres are shown for interacting residues F137, G139 and L142. d, Top view of the neutralized charged ring around the trimeric symmetry axis. e, Side view of the top HR2 with intraprotomeric hydrophobic interactions of L487 (cyan). f,g, Top view around the trimeric symmetry axis (f) and side view (g) of the aromatic cluster showing interactions of both observed 489Y rotamers (cyan). Two different densities were observed for Y489, interacting either with F140 or with both F140 and F137, the N-terminal residue of the fusion peptide. Van der Waals surfaces are shown for the other aromatics. h, Side view (left) of the stabilized HR2 stem and top views (right) with the top layer showing the stabilizing W505 residues (cyan), the middle layer showing the stabilizing F509 residues (cyan) and the bottom layer with L512.

Fig. 6. Immunogenicity and protective efficacy of RSV preF-Δfoldon.

a–d, Balb/c mice were immunized at week 0 and week 4 with formulation buffer (Mock, n = 3) or with AS01_E-adjuvanted preF or preF-Δfoldon protein (see Supplementary Fig. 7 for preF designs) at indicated doses (n = 6 per group). Two weeks after the second immunization, preF-binding antibody titres (a), foldon-binding antibody titres (b) and RSV-A CL57–FFL VNT (c) were measured in all mice; VNTs against RSV-A 18-001989 and RSV-B 17-058221 were measured in the highest dose groups and mock animals (d). Black bars represent the mean response of each group, and dotted lines refer to the LLOD or lower limit of qualification (LLOQ). All measurements under the LLOD and LLOQ were set at the LLOD and LLOQ, respectively. e,f, To assess protective efficacy, Balb/c mice (n = 3 per group) were immunized with the indicated doses of AS01_B-adjuvanted preF or preF-Δfoldon protein or mock control at week 0 and week 4 and intranasally challenged at day 49 with RSV CL57–FFL. Luciferase expression was measured in the nose (e) and lungs (f) before the challenge (day 0) and on days 3, 4, 5, 6, 7 and 10 after the challenge. Means and standard deviations are indicated. The background is indicated with a dotted line. g, Luciferase expression within the animals on day 5. h,i, Immunogenicity of RSV preF-Δfoldon in pre-exposed Balb/c mice. Mice were immunized with unadjuvanted preF or preF-Δfoldon 20 weeks after pre-exposure (n = 7 per group) or were mock immunized (n = 5). Naive mice (n = 3) were added as controls. Serum samples were isolated 6 weeks after immunization. PreF-binding antibody titres (h) and RSV-A CL57–FFL VNT (i) were determined. j,k, Cynomolgus macaques (n = 4) with pre-existing immunity were immunized with 50 µg RSV-A preF-Δfoldon. PreF-binding antibody titres (j) and RSV-A CL57–FFL VNT (k) were determined in serum isolated at the indicated time points. Mean titres (log₂ for VNT and log₁₀ binding IgG titres) with standard deviation are plotted. No LOD and LLOQ are defined. Statistical testing (two sided) was performed across doses using a Tobit model (a–d) or a Tobit model with Dunnett correction for multiple comparisons (h,i). Source data