RSV G peptide immunization and challenge study in mice.

(A) Schematic representation of mouse immunization and challenge schedule. BALB/c mice (N = 6 per group) were immunized i.m. with 25 μg of KLH-conjugated peptides: G 66–90, G 90–110, G 129–152, G 148–178, G 169–207, G 236–263, G 245–274, G 263–298 or with 20 μg of RSV strain A2 REG 67–298 with Emulsigen adjuvant, or with PBS as a control. After the second immunization, blood was collected from the tail veins. Then, mice were challenged intranasally with 106 PFU of RSV rA2-Line19F-FFL. In vivo imaging of lungs was performed daily for 5 days following RSV infection. Mice were sacrificed on day 5 post-challenge, when lungs and blood were collected. (B) Serum samples were collected from individual mice after second immunization and antibodies were analyzed using ELISA plates coated with purified RSV rA2-Line-19F-FFL. The serum samples were serially diluted and the detection of antibodies were measured by absorbance at 490 nm. The mean + SD absorbance for each dilution is indicated. (C) End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an optical density (OD) twice that of the negative control. (D and F) Live whole body imaging was performed daily to detect firefly luciferase activity in the lungs (D) and nasal cavity (F) on day 5 post challenge. Graphs represent the quantification of total flux (photons). 50% and 30% of the mean flux value of the PBS control group is indicated by the two dotted lines. The lowest grey line labeled ‘UC’ represents the average flux values of the uninfected control mice (3.74 x 104 for lungs and 1.48 x 104 for nasal cavity). (E) Lung viral loads at 5 days post-viral challenge as measured by plaque assay. Statistical significance was tested by one-way ANOVA and Bonferroni multiple-comparison tests. ****, p p p p p = 0.1223) (G) and vs. Lung viral loads (r = -0.4835; p = 0.0053) (H) and vs. Nasal Cavity Fluxes (r = -0.0532; p = 0.0.7880) (I).