growth kinetics of all 4 pLAIVs in MDCK cells were related. have been evaluated in phase I clinical tests (4, 22, 23). The development of a robustly immunogenic pLAIV candidate for H5N1 avian influenza viruses has been demanding. An H5N2 pLAIV derived within the Len/17/57 MDV backbone was infectious and immunogenic in 50% of participants, although antibody titers were modest compared to those elicited by seasonal influenza vaccination (23). In contrast, an H5N1 pLAIV formulated with the AA/6/60 MDV was shed inside a minority of participants and was not immunogenic in healthy adults (24). However, both H5 pLAIVs induced long lasting B cell memory space responses, as shown by a rapid and powerful recall response following boost with inactivated H5 vaccine (25, 26). It has been speculated the Len/17/57-centered viruses are more infectious and immunogenic than the AA/6/60-centered vaccine viruses but the basis for the observed variations in viral replication and antibody response MLN8237 (Alisertib) between the AA/6/60- and Len/17/57-derived H5 LAIVs is not known. To directly compare characteristics of H5 pLAIV formulated with each MDV, we generated reassortant viruses by reverse genetics. All vaccine viruses bore surface antigens from your same low pathogenic avian influenza (LPAI) disease, but diverse in internal gene composition to represent the formulations of H5 pLAIV candidates that have been evaluated in clinical studies. We compared the biological characteristics of these viruses and evaluated replication, immunogenicity, and safety from challenge having a heterologous highly pathogenic H5N1 avian influenza (HPAI) disease in the ferret model. Ferrets are naturally susceptible to most influenza A viruses, generate powerful antibody reactions to influenza antigens, and demonstrate medical signs of illness (27) and are therefore a valuable model for pre-clinical evaluation of influenza vaccine candidates. METHODS Cells and viruses 293T and Madin-Darby canine kidney (MDCK) cells were maintained as explained previously (28). MLN8237 (Alisertib) Main human nose epithelial cells (hNEC; PromoCell) and main human being bronchial epithelial cells (NHBE; Lonza) were seeded onto membrane helps (6.5 mm Transwell; Corning, Inc.) coated with collagen type I (Corning, Inc) and managed following COL24A1 manufacturers recommendations. After monolayers reached confluence, an air flow liquid interface (ALI) was founded and cells were differentiated for four weeks before illness. All viruses in this study were propagated in embryonated chicken eggs (Charles River SPAFAS). Viral cDNA of the HA and NA of A/duck/Potsdam/1402-6/1986 (A/dk/Potsdam; H5N2 LPAI) were generated by RT-PCR following isolation of viral RNA. The unmodified hemagglutinin (HA) and neuraminidase (NA) coding sequences were cloned into pAD3000, a derivative of pHW2000 (28). Reassortant attenuated vaccine viruses were generated using an eight plasmid reverse genetics system as previously explained, by co-transfection of 293T and MDCK cells (29). Briefly, the two 6:2 reassortant vaccine viruses were rescued with the six internal protein genes of either Len/17/57 or AA/6/60; the 7:1 vaccine viruses were rescued with only the H5 HA combined with the N2 section from either MDV (Table S1). The 50% cells culture infectious dose (TCID50) was identified as previously explained (14) and titer was determined from the Reed-Muench method (30). Sequences of pLAIV stock viruses were verified by Sanger sequencing (BigDye v3.1, ABI). analysis of H5N2 pLAIVs The phenotype of the recombinant and A/dk/Potsdam viruses was evaluated by plaque assay, as explained previously (29), with duplicate plates incubated in the permissive temp (33C) or the restrictive temp (39C). Results were individually verified by TCID50 assay on MDCK cells. Growth curves were generated by inoculating MDCK cells, NHBE cells, and hNEC cells at a multiplicity of illness (MOI) of 0.01, while described previously (31, 32). Supernatant was sampled at serial time points. The amount of infectious disease in each sample was MLN8237 (Alisertib) determined by TCID50 assay in MDCK cells at 33C. Ferret studies All ferret experiments were authorized by and carried out in compliance with the guidelines of the Institutional Animal Care and Use Committee in the National Institute of Allergy and Infectious Diseases (NIAID) in the National Institutes of Health (NIH). MLN8237 (Alisertib) All study procedures and experiments were carried out in biosafety level 3 facilities in accordance with the Select Agent recommendations.
