The flow-through fraction is affinity purified using lentil lectin washed and eluted from the column with buffer containing methyl-α-d-mannopyranoside (MMP) and polysorbate (PS) 80. The eluted fraction was further purified by cation exchange (sulfate) chromatography. The product was sterile filtered (0.22 μm) and formulated with buffer containing 25 mM sodium phosphate, pH 6.2, 1% histidine, 0.01% PS80. The vaccine was adsorbed to aluminum phosphate (aluminum as phosphate salt in 0.15 M

NaCl without CHIR-99021 purchase buffer) purchased from Brenntag Biosector, Frederikssund, Denmark. Inbred 6–8 weeks Sigmodon hispidus (cotton rats) were obtained from Sigmovir Biosystems, Inc. (Rockville, MD). All studies were conducted in accordance with the NRC Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act and the CDC/NIH Biosafety in Microbiological and Libraries Medical Laboratories under applicable laws and guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC). Lot 100 formalin-inactivated RSV vaccine (FI-RSV) manufactured by Pfizer in mid-1960s [30], and RSV-A Long and RSV-B 18537 were provided by Sigmovir Inc. The RSV–A viruses were PD0332991 ic50 propagated in HEp-2 cells. A pool of virus designated as hRSV-A Long Lot no. 021413 at

approximately 2.0 × 107 plaque forming units (pfu)/ml was stored at −80 °C. RSV-B 18537 (RSV-B) (ATCC, Manassas, VA) was propagated in MA-104 cells. A pool of virus designated as hRSV-B Lot no. 12/03, at approximately 2.7 × 106 pfu/ml 10% was stored at −80 °C. Cotton rats (n = 8) were immunized intramuscularly

(IM) on day 0 and 28 with FI-RSV, RSV-F nanoparticle vaccine with and without Dichloromethane dehalogenase adjuvant, RSV A 1 × 105 pfu intranasally and compared to palivizumab 15 mg/kg given IM, one day prior to challenge. Sera were obtained on day 0, 28, 49 and on day 54 post-challenge. RSV challenge was performed on day 49 intranasally with 1 × 105 pfu in 100 μl (50 μl/nare) RSV-A Long strain and lung tissue collected on day 54. For the dose-descalation active immunization study, cotton rats received two vaccinations of 0.003, 0.03, 0.3, or 3.0 μg RSV F vaccine adjuvanted with aluminum phosphate on Day 0 and Day 21 and compared to palivizumab 5.0, 2.5, 1.25 or 0.625 mg/kg IM on day 41. Sera were obtained on day 0, 21, 42 prior to challenge, on day 46 post-challenge and stored at −20 °C until tested. A pool of immune sera from RSV F nanoparticle vaccine-immunized cotton rats was prepared and assayed in the PCA ELISA as described below. Cotton rats (n = 5/group) were then passively immunized by IM with 0.6, 1.4 or 5.6 mg/kg of palivizumab-like antibody activity and compared to palivizumab given at 5.0, 2.5, 1.25 or 0.625 mg/kg IM on day 41. RSV challenge was performed on day 42 by intranasal administration of 100 μl (50 μl/nare) live RSV-B 18537 (1 × 105 pfu).