Cotton rat immune responses to virus-like particles containing the pre-fusion form of respiratory syncytial virus fusion protein

Abstract

Background: Virus-like particles (VLPs) based on Newcastle disease virus (NDV) core proteins, M and NP, and containing two chimera proteins, F/F and H/G, composed of the respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective anti-RSV neutralizing antibodies in mice. Furthermore, immunization of mice with a VLP containing a F/F chimera protein with modifications previously reported to stabilize the pre-fusion form of the RSV F protein resulted in significantly improved neutralizing antibody titers over VLPs containing the wild type F protein. The goal of this study was to determine if VLPs containing the pre-fusion form of the RSV F protein stimulated protective immune responses in cotton rats, a more RSV permissive animal model than mice.

Methods: Cotton rats were immunized intramuscularly with VLPs containing stabilized pre-fusion F/F chimera protein as well as the H/G chimera protein. The anti-RSV F and RSV G antibody responses were determined by ELISA. Neutralizing antibody titers in sera of immunized animals were determined in plaque reduction assays. Protection of the animals from RSV challenge was assessed. The safety of the VLP vaccine was determined by monitoring lung pathology upon RSV challenge of immunized animals.

Results: The Pre-F/F VLP induced neutralizing titers that were well above minimum levels previously proposed to be required for a successful vaccine and titers significantly higher than those stimulated by RSV infection. In addition, Pre-F/F VLP immunization stimulated higher IgG titers to the soluble pre-fusion F protein than RSV infection. Cotton rats immunized with Pre-F/F VLPs were protected from RSV challenge, and, importantly, the VLP immunization did not result in enhanced respiratory disease upon RSV challenge.

Conclusions: VLPs containing the pre-fusion RSV F protein have characteristics required for a safe, effective RSV vaccine.

Fig. 1

Anti-RSV F protein IgG immune responses to soluble Pre-F and Post-F target antigens. Figure shows titers with time after immunization of anti-F protein IgG in pooled sera that bind to the soluble pre-fusion F protein (a) and that bind to the soluble post-fusion F protein (c). Data shown are the averages of four separate determinations. b and d show titers in sera from individual cotton rats obtained at 49 days post immunization that bind to soluble pre-fusion F protein (b) or that bind to soluble post-fusion F protein (d). b Differences between the RSV group and the VLP groups are significant (p ≤ 0.0001) while the differences between VLPs low and VLPs high are not significant. d Differences between groups are not significant. Titers are defined in “Methods”. Pre-F/F VLPs low and Pre-F/F VLPs high indicate a dose (IM) of 50 and 150 µg total VLP protein/animal, respectively in the prime immunization. Boost immunizations, at day 21, were 17 and 50 μg/animal, respectively. RSV prime and RSV boosts were 1 × 10⁵ pfu/animal (IN)

Fig. 2

Anti-RSV G protein IgG immune responses to soluble RSV G protein. Figure shows titers with time, after immunization with VLP-H/G + Pre-F/F or RSV, of anti-G protein IgG in pooled sera that bind to the soluble G protein. Data are the average of three separate determinations. VLP and RSV immunizations were as in Legend to Fig. 1. Boost immunization was at day 21

Fig. 3

Virus neutralization titers in sera from immunized cotton rats. a Representative data from plaque reduction assays of pooled sera obtained at 49 days post immunization and used to determine neutralization titers. Arrow indicates titer for VLP-low dose. b RSV A2 neutralization titers with time in pooled sera. Data are the average of five separate determinations by the method shown in a. Arrow indicates time of the boost immunization. RSV A2 neutralization titers in sera from individual cotton rats at day 35 (c) and day 49 (d) post immunization, determined as shown in a. Immunogens are shown at the bottom of the panels. Differences between the RSV group and the VLP groups are significant (p = 0.0012 and p = 0.019, respectively). Differences between the two VLP groups are not significant

Fig. 4

Protection of immunized cotton rats from RSV challenge. Panels show titers of virus/gm in lung (left) and nasal (right) tissue four days after RSV A2 challenge of five cotton rats at 49 days post immunization. Immunogens are shown at the bottom for each group of five animals. Statistically significant differences between groups are indicated by p values at the top of each panel

Fig. 5

Assessment of lung pathology after RSV challenge of immunized cotton rats. Panels show scores of lung inflammation in ten cotton rats/group immunized with immunogens indicated at the bottom of each panel and then challenged with RSV at 49 days post immunization. At 4–5 days post challenge, lungs were harvested and tissue sections stained and scored for inflammation as described in "Methods". Differences between FI-RSV immunized animals and all other groups are statistically significant except for scores of perivasculitis, which are not significant. Differences between mock vaccinated, RSV infected, and VLP vaccinated animals in all categories are not statistically significant.

Fig. 6

Lung sections of RSV challenged immunized cotton rats. Panels show representative H&E stained lung sections of cotton rats that were not immunized (a, b), immunized with RSV (c, d), mock immunized (e, f), immunized with low dose Pre-F/F containing VLPs (g, h), or high dose Pre-F/F containing VLPs (i, j), or FI-RSV immunized animals (k, l), all after an RSV challenge at 45 days post immunization. Large arrows indicate patches of inflammatory cells. Small arrowheads indicate cell infiltration into the alveolar space (alveolitis). a, c, e, g, i, k show ×40 magnification while b, d, f, h, j, l show ×100 magnification