Design and Preclinical Evaluation of a Nanoparticle Vaccine against Respiratory Syncytial Virus Based on the Attachment Protein G

Abstract

Human respiratory syncytial virus (RSV) poses a significant human health threat, particularly to infants and the elderly. While efficacious vaccines based on the F protein have recently received market authorization, uncertainties remain regarding the future need for vaccine updates to counteract potential viral drift. The attachment protein G has long been ignored as a vaccine target due to perceived non-essentiality and ineffective neutralization on immortalized cells. Here, we show strong G-based neutralization in fully differentiated human airway epithelial cell (hAEC) cultures that is comparable to F-based neutralization. Next, we designed an RSV vaccine component based on the central conserved domain (CCD) of G fused to self-assembling lumazine synthase (LS) nanoparticles from the thermophile Aquifex aeolicus as a multivalent antigen presentation scaffold. These nanoparticles, characterized by high particle expression and assembly through the introduction of N-linked glycans, showed exceptional thermal and storage stability and elicited potent RSV neutralizing antibodies in a mouse model. In conclusion, our results emphasize the pivotal role of RSV G in the viral lifecycle and culminate in a promising next-generation RSV vaccine candidate characterized by excellent manufacturability and immunogenic properties. This candidate could function independently or synergistically with current F-based vaccines.

Keywords: RSV; attachment protein G; lumazine synthase; nanoparticle; vaccine.

Figure 1

Antigenic overview of RSV G. (A) Schematic of hRSV G with labeled domains. N- and O-linked glycosylation sites are indicated on the ectodomain. Zoom-in between amino acid positions 157 and 198 of the cystine noose is indicated. The epitopes of anti-RSV G monoclonal antibodies CB002.5 and CB017.5 are shown on the CCD amino acid sequence in green and magenta, respectively. CT: cytoplasmic tail, TM: transmembrane, CCD: central conserved domain. (B) RSV A2-GFP reporter gene VNA using anti-RSV F or G monoclonal antibody on A549 cells (left panel) and hAEC cultures (right panel). Infected cells turn green by reporter protein expression. (C–E) Preclinical evaluation of a proof-of-concept RSV G particle-based vaccine. (C) CCD binding antibody titers determined by ELISA using immobilized CCD peptides and displayed as log10 relative potency (rel pot). (D) RSV A neutralizing antibody titers determined by VNA using firefly luciferase (FFL) labeled RSV strain and A549 cells and displayed as log2 of the 90% FFL inhibitory serum dilution (IC90) in serum collected on day 42 from mice (n = 3 or 5) immunized on day 0 and 21 with buffer or with biotinylated G peptide coupled to streptavidin (Strep-G) and adjuvanted with 2% Adjuplex. Bars represent mean responses per group. (E) VNA titers using RSV A expressing a GFP reporter on hAEC cultures with pooled serum samples of Strep-G (C) or preF protein-immunized mice.

Figure 2

RSV G lumazine synthase nanoparticles. (A) Overall structure of self-assembling Aquifex aeolicus lumazine synthase particles. Monomeric subunits oligomerized into a pentamer are indicated by colors. Shades of gray indicate the organization of 12 pentamers forming 60-mer nanoparticles. The zoom-in depicts a single monomer with the locations indicated where peptides can be fused. (B) Analytical SEC traces of AaLS-G particles as determined in cell supernatant. Expression and oligomerization of N-terminal, C-terminal, or internal genetic fusion of RSV G CCD sequences to AaLS. The particle (‘P’) and aggregate (‘A’) peaks are indicated. The different line colors match the construct identifiers in the left panel. (C) Schematic of addition of glycan groups to AaLS-G nanoparticle designs. (D) Expression and oligomerization as assessed by analytical SEC of AaLS particles to which N-linked glycosylation sites were added. The particle (‘P’) and aggregate (‘A’) peaks are indicated.

Figure 3

Characterization of purified lead AaLS-G nanoparticle candidates. (A) SEC-MALS traces of purified AaLS-G nanoparticles. OD280 UV traces appear in blue, and RI mass distribution is overlaid in red. AaLS-G nanoparticles eluted from the column at 4.6 min. (B) NS-TEM microscopy overview images showing the AaLS nanoparticles. Scale bar indicates 50 nm. (C) Particle size determination using dynamic light scatter (DLS). Z-average appears in grey, and the polydispersity index is noted in orange. Average of three measurements are plotted per sample; error bars indicate SD. (D–F) Particle stability in forced degradation experiments. (D) Melting temperature determined using DSF. The first derivative of the fluorescence signal is plotted. The lowest point on the graph corresponds to Tm50 values. (E) Analytical SEC OD280 UV traces of purified AaLS-G nanoparticles after 30 min heat destabilization at indicated temperatures compared to 4 °C control samples. AaLS-G nanoparticles eluted from the column at 3.5 min while other peaks correspond to degradation products. (F) Analytical SEC after indicated number of freeze–thaw cycles. Graph depicts area under the curve of the trimer peak after 1x and 5x freeze–thaw cycles as a trimer percentage relative to the 4 °C control sample. (G) Particle stability after storage of purified AaLS-G nanoparticles at 4 °C for 32 months as analyzed using analytical SEC. As a reference, freshly thawed material stored at −80 °C was used.

Figure 4

Preclinical evaluation of AaLS-G nanoparticle. (A) Experimental design of the mouse immunogenicity study. Mice were immunized with 1 or 10 µg AaLS-G nanoparticles displaying RSV G CCD peptide adjuvanted with 2% Adjuplex or formulation buffer at day 0 and 21 (n = 3 or 5, respectively). (B) RSV G CCD binding IgG antibody titers were determined by ELISA using immobilized CCD peptides and displayed as log10 relative potency (rel pot). (C) RSV A neutralizing antibody titers determined with VNAs using Firefly Luciferase (FFL) labeled RSV A strain CL57 and A549 cells. Titers are displayed as Log2 of the 90% FFL inhibitory serum dilution (IC90) in serum samples isolated at day 20 and/or day 42. Bars represent mean responses per group; the dashed line indicates the lower limit of quantification (LLOQ) of the assay. (D,E) RSV A2-GFP neutralizing antibody titers of serum from 10 µg dosed mice, isolated on day 42 and pooled per immunogen, in hAEC-based VNAs. (D) Depicted are GFP signals of each insert, 96 h post-infection. Infected cells turn green by reporter protein expression. (E) hAEC VNA titers shown as log2 values of the 90% inhibitory concentration (IC90). Bars indicate mean values of two technical replicates depicted by dots.