Nasal wash, BAL, nose Akt inhibitor and lung tissues were collected on Day 46. RSV F-specific antibodies in cotton rat sera were measured in an enzyme linked immunosorbent assay (ELISA) as previously described [25]. Competitive inhibition by cotton rat sera of the binding of palivizumab monoclonal antibody
(ASD Specialty Heath Care Inc., Chicago IL) was measured by an ELISA method as previously described [25]. Serum RSV virus neutralization titers were determined as described previously [25]. Five days after intranasal RSV challenge, cotton rats were sacrificed and the lungs harvested. Lung tissues were homogenized and clarified by centrifugation at 12,000 × g for 10 min. Virus titer in the supernatant was determined by plaque assay as described previously [25]. Lung tissue slides were stained with hematoxylin, eosin (H&E) and observed under a Nikon Eclipse microscope. Slides were evaluated in a blinded fashion using a score of 0–4 (0 = none; 1 = minimal; 2 = mild; 3 = moderate;
4 = maximum inflammation) in order of increasing severity for each of the following 5 parameters: (a) peribronchiolitis; (b) perivasculitis; (c) bronchoiolitis; (d) alveolitis and (e) interstitial pneumonitis as described by Prince et al. [31]. Summary scores for animals in each group were used to generate an overall score/group expressed as the arithmetic mean + SEM of the individual animals. Comparisons between mean scores of each group and non-immune animal challenge scores were analyzed using Student’s t-test. The sum of the scores of five mTOR inhibitor parameters
per animal was used for analysis of histopathology data. Pair wise t-test was analyzed in EXCEL while the GMT and 95% CI were calculated using Graph Pad Prizm. Immune responses to RSV F nanoparticle vaccine (30 μg) administered IM in the presence or absence of adjuvant were compared to animals that received passively transferred palivizumab at the recommended human dose of 15 mg/kg. As controls, animals were infected with 105 pfu of RSV-A Long and allowed to recover, or vaccinated with FI-RSV (Lot 100 at 1:25 dilution), or treated with placebo. Three weeks after the second vaccine dose the immunization with unadjuvanted whatever RSV F nanoparticle vaccine had induced titers of anti-RSV F serum IgG (Fig. 1A) that were significantly higher (p < 0.001, t-test) than cotton rats immunized with FI-RSV antigen or infected with RSV-A virus (p < 0.001, t-test). Adjuvant enhanced RSV F vaccine antibody titers by about 10-fold after the boost ( Fig. 1A). Cotton rats that received palivizumab (IM injection) exhibited lower anti-RSV F IgG serum titers compared to the polyclonal responses obtained following immunization with adjuvanted RSV F (GMT = 1926 vs 1469,084 E.U., respectively Fig. 1A). Antigenic site II on the RSV F polypeptide is the target of palivizumab [32].