The overall strength of the antibody response was assessed by converting the longitudinal data from each animal into an area under the curve (AUC) value and mean AUC values for each group were compared

The overall strength of the antibody response was assessed by converting the longitudinal data from each animal into an area under the curve (AUC) value and mean AUC values for each group were compared. Fluzone?+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and Sirt2 plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine. for 10 min, and all but 1 ml of the supernatant was removed and stored at ?80C. The frozen aliquots were subsequently used to determine infectious virus titer. The 1 ml aliquot of fresh supernatant was immediately processed for RNA isolation to assess viral RNA (vRNA) levels. 2.6. Influenza virus RNA PCR To determine the amount of virion-associated RNA in respiratory secretions, fresh tracheal lavages were processed and quantified D-(+)-Phenyllactic acid by RT-PCR as previously described [22]. Briefly, 1 ml of fresh tracheal lavage was centrifuged at 1,000 g for 10 min. The supernatant was removed and lysed in TRIzol LS (Invitrogen Life Technologies). cDNA was prepared using random hexamer primers (Amersham Biosciences) and SuperScript III reverse transcriptase (Invitrogen Life Technologies). The Influenza A virus matrix gene in the samples was quantified using a D-(+)-Phenyllactic acid real-time RT-PCR assay on a Prism 7900 sequence detection system (Applied Biosystems) and previously described influenza A virus matrix gene-specific PCR primers [28]. D-(+)-Phenyllactic acid The influenza A matrix copy numbers were determined by a modification of a method previously described [29]. Briefly, the copy number of matrix gene was determined by interpolation of the average measured threshold D-(+)-Phenyllactic acid cycle number onto a standard curve produced with a purified plasmid containing a fragment of the M1 gene cloned from the A/Memphis/7/2001 stock. Quantification of the purified plasmid was based on A260 measurements. 2.7. Influenza Antibody ELISA Titers of anti-influenza antibodies were determined by a modification of a method previously described [22, 30]. Briefly, all plasma, tracheal aspirate, and nasopharyngeal secretion samples were initially tested for anti-A/New Caledonia/20/99 in a screening assay. The screening dilutions for IgG and IgA in plasma were 1:800 and 1:80, respectively, and all secretion screening dilutions were 1:4 for both IgG and IgA. Results of the screening assay were calculated from optical density absorbance units (OD) using the following ratio: change in OD (OD)/cutoff, where OD is defined as the difference between the mean OD of a diluted sample tested in two influenza Ag-coated wells and the mean OD of the same diluted sample tested in two uncoated wells. The cutoff value is the mean OD of two pre-treatment time points of a sample plus 3 SD values. If only one pre-treatment time point was available, the cutoff value is the mean OD of one pre-infection time point run in two independent assays plus 3 SD values. If the OD/cutoff ratio for a sample was 1.0 and the OD 0.10, the sample was considered to be positive and the titer of anti-influenza antibody was determined. To determine anti-influenza A antibody titers in plasma or secretion samples that were positive in the screening assay serial doubling dilutions of the samples were loaded onto a 96-well plate (Nunc-Immuno Maxisorp plate II) with uncoated wells and wells coated with detergent disrupted A/New Caledonia/20/99 influenza A (Biodesign International). Antibody binding was detected with a peroxidase-conjugated goat anti-monkey IgG (Fc) or IgA (Fc) (Accurate Chemicals). For each sample, the endpoint titer of anti-influenza A antibody was defined as the last dilution giving a OD value 0.10. 2.8. HI assay Titers of anti-H1 antibodies were calculated using the revised World Health Organization HI test of hemagglutinin inhibition as previously described [22, 31]. The viral antigen used in the HI was the A/Memphis/7/2001 stock grown in 10-day-old embroynated chicken eggs (Charles River). 2.9. Intracellular cytokine staining for assessing influenza-specific T cell responses For intracellular staining to detect influenza-specific T cells in PBMCs, modifications of previously reported methods were used [22, 32, 33]. Cryopreserved cells collected prior to challenge were stimulated with pediatric Fluzone? at 5g H1/ml. Data were acquired using a FACSAria flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star) and a Mac Pro computer (Apple). At least 100,000 events in the forward/side scatter lymphocyte gate were acquired. The background level of cytokine staining varied from sample to.