Immunogenicity and protection from viral challenge of pre-RSV + GLA-SE in young, seronegative mice.

<p>Young (7 weeks old) BALB/c mice (n = 5 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F pre- and post-conformation at 0.3 μg +/- GLA-SE (2.5 μg/2%) adjuvant. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. (A) Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. (B) 4 days post-challenge (day 39), splenocytes were isolated and stimulated for 24 h with RSV F specific CD4 T-cell and CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. (C) Lung and (D) nasal turbinates viral loads were measured by plaque assay. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Lung T cell profile after RSV challenge in young and aged seronegative mice immunized with post-RSV F formulations.

<p>Young (7 weeks old) and aged (18 months old) BALB/c mice (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 with buffer alone, post-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. 4 days post-challenge (day 39), lung cells were isolated and stimulated with RSV F peptide pool to evaluate intracellular cytokine expression by flow cytometry. Cells were surface stained for CD3 and CD8, intracellularly stained for IFNγ, TNFα, and IL-2, and analyzed on an LSR II for the frequency of responding (A) CD4+ and (B) CD8+ T cells. (C) The percentage of F85-93-pentamer+ CD8 T cells was determined by flow cytometry. (D) The percentage of lung eosinophils was determined by flow cytometry. For (C) and (D), the mean of individual values +/- SD is shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Immunogenicity of post-F and pre-F in aged seronegative mice.

<p>Aged (18 months old) BALB/c mice (n = 6 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F (pre- or post- conformation) at the indicated doses +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. 4 days post-RSV A2 challenge, splenocytes were isolated and stimulated for 24 h with peptides representing <b>(B)</b> RSV F specific CD4 T cell and <b>(C)</b> CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Protection from RSV A2 challenge in aged seronegative mice, immunized with pre- and post-RSV F.

<p>Aged (18 months old) BALB/c mice (n = 6 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F (pre- or post- conformation) at the indicated doses +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. 4 days post-RSV A2 challenge, <b>(A)</b> lung and <b>(B)</b> nasal turbinates viral loads were measured by plaque assay. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Lung T cell profile of aged seropositive mice following immunization with different RSV F formulations and post challenge with RSV A2.

<p>12 months old mice were i.n. immunized with RSV A2 (10⁶ PFU) at day 0 and RSV B (10⁵ PFU) at day 60. Aged seropositive mice were then immunized i.m. at day 176 (18 months old) with buffer alone, post-F or pre-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. At day 190, aged seropositive mice were challenged with RSV A2 (10⁶ PFU). 4 days later, lung cells were isolated and stimulated with RSV F peptide pool. Cells were then surface stained for CD3 and CD8, intracellularly stained for IFNγ, TNFα, and IL-2, and analyzed on an LSR II for the frequency of responding <b>(A)</b> CD4+ and <b>(B)</b> CD8+ T cells.</p

NAb induction of pre-F and post-F formulations in aged seropositive mice.

<p>12 months old mice were i.n. immunized with RSV A2 (10⁶ PFU) at day 0 and RSV B (10⁵ PFU) at day 60. Aged seropositive mice were then immunized i.m. at day 176 (18 months old) with buffer alone, post-F or pre-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. <b>(A)</b> Bleeds were collected at day 50, 120, and 174 to assess the levels of RSV A2 neutralizing antibodies in seropositive animals prior to immunization (baseline). Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. <b>(B)</b> 14 days post immunization, RSV A2 neutralization titers were evaluated. Group means ± SD of individual mice are shown. For statistical analyses, multiplicity adjusted ANOVA test was performed against baseline as a control group, followed by pairwise testing of post-F versus pre-F, * for P<0.05.</p

Immunogenicity of post- RSV F formulations in young and old seronegative mice.

<p>Young (7 weeks old) and aged (18 months old) BALB/c mice (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 with buffer alone, post-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. (A) Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. At sacrifice (day 39), (B) RSV F specific anti-IgG1 and anti-IgG2a were measured by ELISA, and splenocytes were isolated and stimulated for 24 h with peptides representing (C) RSV F specific CD4 T-cell and (D) CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p