The RSV A2 strain was originally gifted from Dr. Barney Graham. Hep-2 cells were obtained from American Type Culture Collection and maintained in DMEM media. The expression plasmid encoding a full-length human codon -optimized RSV A2 F within the pcDNA3 plasmid was described in previous studies (1819). F-VLP vaccine was produced in insect cells by co- expressing RSV F and influenza virus matrix 1 (M1) core protein and characterized as previously described (20). Briefly, Sf9 insect cells were co-transfected with recombinant baculoviruses expressing M1 and RSV F protein in serum-free SF900-II medium. The culture supernatants were collected by centrifugation (6,000 rpm, 20 min) to remove insect cells. The cleared supernatants containing F-VLPs were purified by ultracentrifugation. The purified F-VLPs were resuspended in PBS. F-DNA vaccine was purified using Qiagen Plasmid Mega Kit (Qiagen, Hilden, Germany). RSV was grown in HEp-2 cells, inactivated with formalin (1:4,000 vol/vol) for 3 days at 37°C, purified using ultracentrifugation to remove cell culture supernatants and cellular proteins, and finally confirmed by a modified immuno-plaque assay as previously described (20). Briefly, the infected Hep-2 cell plates were incubated at 37°C, 5% CO₂ for 16 h and then viral supernatants were removed. After overlay with a mixture of 1% agarose and DMEM media, the infected Hep-2 cell plates were further incubated for 3 days, the infected Hep-2 cell plates were fixed with 10% formalin and then overlay agarose was removed. Viral plaques by RSV A2 were immune-stained with 131-2a monoclonal Ab and DAB substrate (Invitrogen, Carlsbad, CA, USA).

For F-VLP and F-DNA vaccine groups, BALB/c mice (6 to 8 wk old, n=5, duplicate) were intramuscularly primed and boosted with 20 μg F-VLP or 100 μg plasmid F-DNA at 0, 3 wk. Second boost vaccination was followed with a half dose of F-VLP (10 μg) or F-DNA (50 μg) respectively at wk 6. The FI-RSV control group was intramuscularly primed (wk 0) and boosted (wk 3) with FI-RSV (2 μg) in alum (50 µg) adjuvant. Each group of immunization and challenge experiments was duplicated. Naïve or vaccinated mice were challenged intranasally with RSV A2 strain (3.3×10⁵ PFU, plaque-forming units) 20 wk after 2nd boost. The individual lungs and bronchoalveolar lavage fluid (BALF) samples were collected day 5 post-challenge to determine inflammatory cellular responses (1718). All animal experiments were approved by the Georgia State University Institutional Animal Care and Use Committees (IACUC A18001) and performed in accordance with relevant guidelines and regulations.

RSV specific Abs (IgG, IgG1, and IgG2a) were determined by ELISA using RSV F protein antigen (BEI) and presented in concentrations based on standard IgG and isotypes as described (17). Neutralizing Ab titers in mouse sera were measured using the red fluorescent RSV A2-K-line19F (200 PFU per well) and lung viral titers by an immunoplaque assay as described (2122).

Individual lungs were immersed in 10% neutral buffered formalin for 24 h, embedded in paraffin, sectioned, and stained with H&E, periodic acid-Schiff stain (PAS) or hematoxylin and congo red (H&CR) as described (222324). For numerical assessment of histopathology and pneumonia in lung tissues, the bronchioles, vessels and interstitial space were initially scored on a scale of 0–3 by blinded observers with the severity scoring system as previously described (2325). A score 1 was assigned when the epithelial lines, vessels, and interstitial surrounding spaces have few infiltrating cells, a score 2 when the epithelial lines and surrounding spaces contain focal aggregates of infiltrating cells or the structure is cuffed by one definite layer of infiltrating cells, and a score 3 when structure is obviously cuffed by two or more definite layers of infiltrating cells with focal aggregates.

BALFs were harvested from the lung airways by infusion with PBS via trachea using a 25-gauge catheter (26). Lung tissues were homogenized, passed through a cell strainer, and spun on 44/67% Percoll gradients to collect lung cells. Cellular phenotypes were determined by flow cytometric analysis using cell surface marker Abs specific for CD3, CD4, CD8, CD11b, CD11c, CD45, F4/80, Siglec F, DX5, and Ly6c (eBioscience, San Diego, CA, USA or BD Pharmingen, San Jose, CA, USA) as previously described (172728). Cells from BALF and lungs were stimulated with the synthetic F_85-93 (KYKNAVTEL) peptide (29) for CD8 T cell activation. Intracellular cytokine-producing cells stained with monoclonal IFN-γ and TNF-α and cell phenotypic marker Abs were acquired by the Becton-Dickinson LSR-II/Fortessa flow cytometer and data analyzed by Flowjo software (Tree Star Inc., Ashland, OR, USA). In the determination of cellular phenotypes, the fraction (%) of each cell phenotypes obtained by flow cytometry was multiplied by the total cell numbers counted in the cell preparations from the lung tissues and BALF.

Results are expressed as mean±SEM; statistical significance was calculated by one-way ANOVA, followed by Tukey's Multiple Comparison test using GraphPad Prism software (GraphPad Inc., La Jolla, CA, USA). A p-value less than 0.05 was considered significant.

To compare the immunogenicity of exogenous F-VLP and endogenous F-DNA vaccine platforms, RSV F protein antigen-specific Abs were determined in immune sera from immunized mice (Fig. 1). Prime dose of F-DNA vaccine induced low levels of RSV F specific IgG Abs, requiring multiple immunizations. Whereas, the F-VLP group of mice induced substantial levels of IgG and IgG2a isotype Abs specific for RSV F protein after prime immunization. Significant levels of RSV F protein specific IgG and IgG2a Abs were induced after second boost with F-DNA vaccine although they were lower than those induced by F-VLP boost (Fig. 1A-C). Both F-VLP and F-DNA vaccines preferentially raised IgG2a isotype dominant Ab responses (Fig. 1B) as evidenced by high IgG2a/IgG1 ratios of Ab responses (Fig. 1D). In contrast, FI-RSV vaccination induced highest levels of IgG1 isotype Abs (Fig. 1C), suggesting Th2 immune responses. RSV neutralizing activity as measured by reduction percentages of fluorescent RSV virus infection and plaque assays was observed at higher levels in both immune sera from the F-VLP and F-DNA groups compared to naïve sera (Fig. 1E). Immune sera of alum adjuvanted FI-RSV vaccinated mice exhibited highest levels of RSV neutralizing activity (Fig. 1E).

To assess the protective efficacy of F-VLP and F-DNA vaccines, immunized mice were challenged with RSV A2 at 20 wk after second boost. F-VLP immunized mice did not show body weight loss, but approximately 10% body weight loss was observed in the naïve mice infected with RSV (Naïve inf, Fig. 2A). In contrast, F-DNA immunized mice displayed significant weight loss up to 15% (Fig. 2A). The highest RSV titers in lungs were detected in unvaccinated naïve mice on day 5 post infection (Fig. 2B). Approximately 10-fold lower lung RSV titers were observed in F-DNA immune mice compared to naïve infection mice, significantly clearing lung viral loads. The F-VLP group lowered lung viral titers by over 200 folds near to the detection limit (Fig. 2B). The alum adjuvanted FI-RSV group effectively cleared lung viral loads below the detection limit (Fig. 2B).

The lung tissue sections were examined by staining with H&E, PAS, or H&CR at 5 days after RSV challenge (Figs. 3 and 4). As expected, mice that were immunized with FI-RSV showed the highest degree of pulmonary histopathology. After RSV challenge, F-DNA vaccination was found to induce significant infiltrates in the interstitial spaces of the lungs, which were similar to or slightly lower than FI-RSV (Fig. 3A and B). Moderate levels of infiltrates were observed in the blood vessels and airways from F-DNA immune mice (Fig. 3D). Meanwhile unvaccinated naïve mice exhibited moderately higher levels of infiltrating histopathology in the interstitial spaces after RSV infection (Fig. 3A and B). F-VLP immune mice did not show overt histopathology infiltrates upon RSV infection.

Mucus production and eosinophilia are also considered RSV disease parameters. FI-RSV immunization showed the highest levels of PAS positive mucus production and H&CR positive eosinophils upon RSV challenge (Fig. 4). F-DNA immunization caused low levels of eosinophils (Fig. 4B and D). The naïve and F-VLP groups did not display PAS positive spots and eosinophils in the lung sections (Fig. 4A and C)

Cellular phenotypes in lung (Fig. 5) and BALF (Fig. 6) were analyzed by flow cytometry using cell type-specific marker Abs at 5 days after RSV challenge. Alum-adjuvanted FI-RSV immune mice showed the highest levels of eosinophils (CD11b⁺CD11c⁻SiglecF⁺) in lung and BALF samples as well as plasmacytoid dendritic cells (pDC, B220⁺CD11c⁺F4/80⁻) in BALF. F-DNA immunized mice displayed higher levels of NK cells (CD3⁻DX5⁺), monocytes (CD11b⁺F4/80⁺Ly6C^high), eosinophils, and pDC in the lungs compared to F-VLP or naïve infection mice. Particularly, eosinophil and monocyte populations were increased by approximately 3-fold in F-DNA mice than those in F-VLP mice. We also observed increased levels in monocytes and pDCs in airway BALF samples from F-DNA immunized mice, compared to those in F VLP (Fig. 6).

We determined the levels of CD8 T cells producing IFN-γ and/or TNF-α cytokines in the lungs at 5 days after RSV challenge by a flow cytometry assay of intracellular cytokine staining. Lung cells from F-DNA immune mice showed significantly higher levels of RSV F_85-93 specific IFN-γ or TNF-α positive CD8 T cells than F-VLP, FI-RSV immune or naïve infection mice (Fig. 5E and F).

Germinal center (GC) phenotypic B cells (IgD⁻B220⁺GL7⁺) in the mediastinal draining lymph node (MLN) were also analyzed by flow cytometry day 5 post challenge. F-VLP immunized mice showed significantly higher levels of GC phenotypic B cells in line with increased levels of serum IgG Abs compared to F-DNA immunization. The F VLP group showed certain levels of IgA Abs in BALF but not the F-DNA group, but overall the IgA Ab levels were very low and no significant levels of IgM Ab levels were detected (data not shown).