Virus-like Particle Vaccines Containing RSV Fusion Protein Cleavage Mutations Are Immunogenic and Protective in Cotton Rats With a Prime Dose

Figures

• 

  Figure 1. Development of RSV mutant F constructs and open trimeric RSV mutant F VLP structure.

• 

  Figure 2. Design and antigenic profiles of mutant RSV F proteins in VLP with pre-F and post-F conformation dependent mAb reactivities. (A) Diagram and (B) amino sequences of RSV mutant F gene constructs to be presented on VLPs. The furin cleavage sites I and II (black arrowheads) divide the F0 protein into F1 and F2 in wild type RSV F gene, F-TMrsv (WT). ∆FP has deletion of the first 8 amino acids of the FP following the furin cleavage site I. F1dm-G4TMpr8 gene included furin cleavage site I (KKRKRR → KKQKQQ) cleavage mutations, marked in dotted arrowhead, and addition of GCN4 before TM domain of influenza PR8 HA (TMpr8). RSV F VLP Ags were coated on 96-well plates. (C) The 1 ng/ml of 131-2A mAb specific for site I epitope. (D) The 1 ng/ml of palivizumab specific for the site II epitope, (E) 5C4 mAb specific for pre-F site Ø epitope, (F) The 1 ng/ml of D25 mAb specific for pre-F site Ø epitope. p27, excised peptide.

• 

  Figure 3. Serum and cell culture IgG Abs from immune cotton rat after prime vaccination with recombinant F protein VLP. Levels of IgG Abs specific for (A) DS-Cav1, (B) post-F, and (C) FI-RSV at 3 wk after prime immunization. RSV-F protein (BEI) specific IgG Abs secreted into in vitro cell culture supernatants from (D) BM, (E) spleen, and (F) MLN at 5-day post RSV A2 infection. Statistical analysis was performed using two-way ANOVA and Dunnett’s or one-way ANOVA and Tukey’s post-multiple comparison tests. All results were shown in mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

• 

  Figure 4. RSV neutralizing activities, lung viral clearance in cotton rats after single dose vaccination. For neutralizing assay, immune sera were inactivated and serially diluted. The diluted sera (n=5 per group) were incubated with RSVA2-K-line19F (500 PFU), then were added to Hep2 cell monolayers for 3 days to determine percentages of plaque reduction and fluorescent images in a condition with 40, 160 and 640-fold diluted sera. (A) A representative fluorescent photography and (B) cell numbers of Hep2 cells infected with RSV A2-K-line19F expressing Katushka 2 fluorescent protein after incubation with immune sera. (C) Neutralizing activity of the serial diluted single dose immune sera. (D) Lung viral titers were determined from individual cotton rat at 5 days after challenge (n=5 per group). Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests. All results were shown in mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

• 

  Figure 5. Cotton rats with F1dG4TMpr8 VLP vaccination display higher levels of IFN-γ cytokine in draining lymph nodes without pulmonary histopathology after RSV challenge. (A) Bronchiolar mucus expression was stained with PAS. (B) PAS positive area (%) by mucus production as scored from 5 individual airways of each cotton rat. (C) Histopathology scores. Histopathology levels on H&E-stained tissue section were scored in the airways, blood vessels, and interstitial spaces on a scale of 0 to 3 according to diagnostic criteria. (D) Draining lymph nodes cells were collected from immunized or naïve cotton rats (n=5 per group) at day 5 after challenge and cultured by stimulating RSV F protein on the plates for 72 h then the levels of IFN-γ and IL-4 were determined by cytokine ELISA. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple comparison tests. All results were shown in mean ± SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

• 

  Figure 6. Cotton rats with F1dG4TMpr8 VLP vaccination display lowest cellular infiltrates in BALF and lung cells after RSV challenge. Cellular distribution proportions of the gates 1, 2, and 3 as quantified by forward (size) and side (granularity) scatter characteristics in flow cytometry analyses of BALF and lung cells. Cellularity of the gate 1 (small lymphocyte-like cells), the gate 2 (large monocytes, neutrophils, and eosinophil-like cells), and the gate 3 (dendritic, granular/myeloid and macrophage-like cells). Cellularity was presented from the results of (A) total BALF and (B) lung cell numbers per cotton rat multiplied by percentages of each population. FSC parameters, proportional to cell cross-sectional area, were used to determine the relative size of individual cells, while SSC parameters, indicative of internal structural complexity and granularity, enabled discrimination of cell populations with differing cytoplasmic density and granule content. Gating strategies for flow cytometry–based analysis of BALF and lung cells, used to identify major population subsets, are shown at the end of each row. Statistical analysis was performed using one-way ANOVA and Tukey’s post-multiple…