Prefusion F-Based Polyanhydride Nanovaccine Induces Both Humoral and Cell-Mediated Immunity Resulting in Long-Lasting Protection against Respiratory Syncytial Virus

Abstract

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in both young children and in older adults. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is currently available. We have developed a novel RSV vaccine composed of a prefusion-stabilized variant of the fusion (F) protein (DS-Cav1) and a CpG oligodeoxynucleotide adjuvant encapsulated within polyanhydride nanoparticles, termed RSVNanoVax. A prime-boost intranasal administration of RSVNanoVax in BALB/c mice significantly alleviated weight loss and pulmonary dysfunction in response to an RSV challenge, with protection maintained up to at least 6 mo postvaccination. In addition, vaccinated mice exhibited rapid viral clearance in the lungs as early as 2 d after RSV infection in both inbred and outbred populations. Vaccination induced tissue-resident memory CD4 and CD8 T cells in the lungs, as well as RSV F-directed neutralizing Abs. Based on the robust immune response elicited and the high level of durable protection observed, our prefusion RSV F nanovaccine is a promising new RSV vaccine candidate.

Figure 1.. Prime-boost vaccination with RSVNanoVax protects against RSV-induced weight loss and pulmonary dysfunction.

BALB/c mice were primed i.n. on day 0 and boosted with 500 μg on day 28. All mice were challenged with 4.8 × 10⁶ PFU RSV-A2 on day 56 (A), day 100 (B), or 6 months (C) post-prime and assessed for weight loss and pulmonary dysfunction as measured by baseline changes in Penh and EF50. Asterisks represent significance between no vaccine and RSVNanoVax and pound symbols represent significance between CpGNanoVax and RSVNanoVax as determined by 2-way ANOVA with a Dunnett’s post hoc test. *^/# p<0.05, **^/## p<0.01, ***^/### p<0.001. No vaccine mice were administered PBS i.n. at both the prime and boost. Data represent mean ± SEM of 2–3 independent experiments (n=8–12).

Figure 2.. Prime-boost RSVNanoVax immunization reduces infectious RSV particles in the lungs.

BALB/c mice were primed i.n. on day 0 and boosted with 500μg on day 28. On day 56 (A), day 100 (B), or 6 months (C) post-prime mice were challenged with 4.8 × 10⁶ PFU RSV-A2 and infectious viral pfu were quantified in the lung on day 4 post-infection. (D) Vaccinated mice were challenged with 1.1 × 10⁶ PFU RSV line 19 on day 56 and infectious viral pfu were quantified in the lung on day 4 post-infection. Statistical significance was determined by one-way ANOVA with a Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. No vaccine mice were administered PBS i.n. at both the prime and boost. RSV immune mice received 4.8 × 10⁶ PFU RSV-A2 at the prime and PBS i.n. at the boost. Data represent mean ± SEM of 2 independent experiments (n=7–10). The horizontal dashed line represents the limit of detection.

Figure 3.. Prime-boost RSVNanoVax vaccination induces RSV-specific systemic and local antibody responses.

Serum was collected from WT mice that received a prime and boost of the indicated nanoparticle formulation, naïve mice, or RSV immune mice that received 4.8 × 10⁶ PFU RSV-A2 56 days or 6 months prior. (A) PreF or (B) postF-directed total IgG, IgG1, or IgG2a was measured by antibody ELISA. (C) F-specific IgA was measured in either whole lung homogenates (preF and postF) or nasal wash fluid (preF only) on day 56 by antibody ELISA. Statistical significance was determined by a 2-way ANOVA with a Dunnett’s post hoc test. Asterisks represent significance between RSVNanoVax d56 and RSV immune d56 and pound symbols represent significance between RSVNanoVax 6 month and RSV immune 6 month. *^/# p<0.05, **^/## p<0.01, ***^/### p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8–10).

Figure 4.. Prime-boost RSVNanoVax vaccination induces RSV-specific serum antibodies with neutralizing capability.

Serum was collected from WT mice that received a prime and boost of the indicated nanoparticle formulation, naïve mice, or RSV immune mice that received 4.8 × 10⁶ PFU RSV-A2 56 days or 6 months prior. (A-C) RSVNanoVax RSV F site-specific competitive binding was determined by competition antibody ELISA. Competition with (A) D25 or (B) motavizumab for binding to preF or competition with (C) motavizumab for binding to postF. Competitive titers are expressed as log2 of the serum dilution that resulted in 50% inhibition of D25 or motavizumab monoclonal antibody binding to the RSV F protein. (D) Neutralizing capacity of serum antibodies was determined by RSV-A2 plaque reduction on Vero cells. Neutralizing titers are expressed as log2 of the serum dilution that resulted in 50% inhibition of viral plaques. Statistical significance was determined by a one-way ANOVA with a Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8–11). The horizontal dashed line represents the limit of detection.

Figure 5.. Antigen-experienced tissue-resident memory CD4 and CD8 T cells are induced by prime-boost RSVNanoVax immunization.

BALB/c mice were primed with 500 μg of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 μg i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8 × 10⁶ PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. Lungs and spleens were harvested on day 42 and analyzed by flow cytometry. Frequency of (A) antigen-experienced (CD11a^hiCD49d⁺) CD4 T cells and (B) antigen-experienced (CD11a^hiCD8^lo) CD8 T cells. Total number of antigen-experienced (C) CD4 T cells and (D) CD8 T cells in the lung. Number of antigen-experienced CD45 intravascular antibody negative (i.v.⁻) (E) CD103⁻CD69⁺ CD4 T cells and (F) CD103⁺CD69⁺ CD8 T cells. Statistical significance was determined by (A and B) 2-way ANOVA with a Tukey’s post hoc test or (C-F) one-way ANOVA with a Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8).

Figure 6.. Prime-boost RSVNanoVax immunization induces RSV-specific functional CD4 and CD8 T cells in the lungs.

BALB/c mice were primed with 500 μg of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 μg i.n. on day 28. No vaccine mice were administered PBS i.n. on both prime and boost days. RSV immune mice received 4.8 × 10⁶ PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. Lungs were harvested on day 42 and analyzed by flow cytometry. Number of (A) antigen-experienced F_85–93 tetramer⁺ CD8 T cells and (B) i.v.⁻ antigen-experienced F_85–93 tetramer⁺ CD8 T cells. Number of antigen-experienced IFN-γ⁺ (C) CD4 and (D) CD8 T cells following stimulation with PMA and ionomycin. Statistical significance was determined by one-way ANOVA (A-D) with a Tukey’s post hoc test. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8).

Figure 7.. Protection afforded by RSVNanoVax is partially mediated by systemic antibodies.

Serum was collected on day 56 from WT donor mice that received a prime and boost of the indicated nanoparticle formulation, naïve mice, or RSV immune mice that received 4.8 × 10⁶ PFU RSV-A2 56 days prior. Recipient BALB/c mice were administered 200 μL serum i.p.. At 24 hours all mice were challenged with 4.8 × 10⁶ PFU RSV-A2 i.n. (A-C) Groups were monitored daily for (A) weight loss, (B) Penh, and (C) EF50. Asterisks represent significance between no vaccine and RSVNanoVax and pound symbols represent significance between CpGNanoVax and RSVNanoVax as determined by 2-way ANOVA with a Dunnett’s post hoc test. (D) Infectious viral PFU were quantified in the lung on day 4 post-infection by plaque assay. Statistical significance was determined by one-way ANOVA with a Tukey’s post hoc test. *^/# p<0.05, **^/## p<0.01, ***^/### p<0.001. Data represent mean ± SEM of 2 independent experiments (n=9). The horizontal dashed line represents the limit of detection.

Figure 8.. Prime-boost RSVNanoVax vaccination mediates viral clearance in an outbred population.

Swiss Webster mice were primed with 500 μg of the indicated nanoparticle formulation i.n. on day 0, and boosted with 500 μg i.n. on day 28. No vaccine mice were administered PBS i.n. at both the prime and boost. RSV immune mice received 4.8 × 10⁶ PFU RSV-A2 i.n. at the prime and PBS i.n. at the boost. All mice were challenged with 4.8 × 10⁶ PFU RSV-A2 on day 56. Infectious viral PFU were quantified in the lung on day 2 or 4 post-infection by plaque assay. Statistical significance was determined by 2-way ANOVA with a Tukey’s post hoc test. ***p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8–10). The horizontal dashed line represents the limit of detection.

Figure 9.. Prime-boost RSVNanoVax vaccination induces systemic RSV-specific antibodies in an outbred population.

Serum was collected from Swiss Webster mice that received a prime and boost of the indicated nanoparticle formulation, naïve mice, or RSV immune mice that received 4.8 × 10⁶ PFU RSV-A2 56 days or 6 months prior. (A) PreF or (B) postF-directed total IgG, IgG1, or IgG2a antibodies were measured by antibody ELISA. Statistical significance was determined by 2-way ANOVA with a Tukey’s post hoc test. Asterisks represent significance between RSVNanoVax and RSV immune. *p<0.05, **p<0.01, ***p<0.001. Data represent mean ± SEM of 2 independent experiments (n=8–10).