Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses

Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses. NA-MNT is a reliable and high throughput method which could facilitate the development of candidate pandemic influenza vaccine. == Introduction == Newly emerged avian influenza A viruses have a significantly negative impact on public health. Specifically, the highly pathogenic avian influenza H5N1 virus has infected 860 humans, with a mortality rate of 52%, according to the World Health Organization [1], whereas H7N9, first emerged in 2013, has infected over 1500 humans and caused 612 deaths TAK-438 (vonoprazan) [2]. Effective vaccines against these viruses in humans are urgently needed. As an important part of vaccine development, standard and reliable methods with high-throughput capacity are needed to evaluate the immune response elicited by influenza vaccines. Several candidate assays, including neutralization and hemagglutination TAK-438 (vonoprazan) inhibition (HI) assays, have been used to assess the efficacy of the influenza vaccines. The microneutralization test (MNT) has proven to be useful in evaluating the immunogenicity of pandemic influenza H5N1 or H7N9 vaccines [36], as well as determining the prevalence of H5N1 in human populations that have had contact with infected birds, given that it measures the neutralizing activities of TAK-438 (vonoprazan) antibodies with greater sensitivity than the traditional HI assays [3,4,7]. The MNTs have a similar neutralization step, in which sensitive cells are inoculated with a mixture of viruses and serum, but have different final steps as read out including microscopically observing cytopathic effects (CPEs), assessing virus hemagglutination with red blood cells, or detecting viral proteins by ELISA. Using the CPE method, some influenza viruses induce uncharacteristic CPEs, which makes view of the results subjective and dependent on the encounter of the observer. Although ELISA-MNT is definitely more sensitive than HI as it quantifies viral nucleoproteins by ELISA, it is a relatively long process with multiple methods, making it challenging for assay standardization. Indeed, considerable variations have been observed in inter-laboratory assessment studies TAK-438 (vonoprazan) [6,8]. In this study, we describe an MNT that actions neuraminidase (NA) activity as the readout (NA-MNT). Only lysates from cells infected with disease and NA substrates are needed. This simple method was used here to measure antibody titers induced by pandemic influenza vaccines in comparison with results generated by both HA assay and ELISA-MNT. == Materials and methods == Rabbit Polyclonal to TSN == Cells and viral strains == MDCK (Madin-Darby canine kidney) cells were cultured in Dulbeccos revised essential medium (DMEM) comprising 1% penicillin/streptomycin (Gibco Existence Technologies, Grand Island, NY, USA), 10% fetal calf serum (Hyclone South logan, UT, USA) and 1% L-glutamine and managed inside a 5% CO2 incubator at 37C. The viruses tested were vaccine strains from The National Institute for Biological Requirements and Control (NIBSC; Potters Barr, UK), including A/Vietnam/1194/2004 NIBRG-14 (H5N1) and A/Anhui/01/2013 NIBRG-268 (H7N9). All viruses were cultivated in 10-day-old embryonated chicken eggs for 4896 h at 35C. Cellular debris from harvested allantoic fluids was eliminated by centrifugation at 3000 rpm for 10 min, with the harvested viral aliquots becoming stored at 70C. The infectious titers of the viral stocks were identified as explained previously [9]. The 50% cells culture infective dose (TCID50) was determined using ReedMuench method. == NA assays == NA assays were performed as explained previously [10] using the substrate 2-O-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MU-NANA) (Sigma-Aldrich, St Louis, MO, USA). Cleavage of MU-NANA by NA releases fluorescent methylumbelliferone which is definitely consequently quantified using a fluorescence plate reader. == Human being serum samples == A panel of serum samples was collected from 40 volunteers who received two doses of inactivated H5N1 influenza vaccine (A/Vietnam/1194/2004 NIBRG-14, 15 g HA/dose), kindly provided by Wuhan Institute of Biological Products Co., Ltd, Wuhan, China. The trial was authorized by the Honest Committee of Jiangsu Provincial Center for Disease Control and Prevention and authorized atchinadrugtrials.gov.cn(ID: CTR20131644). Serum samples were also collected from human subjects vaccinated with H7N9 influenza vaccine (A/Anhui/01/ 2013 NIBRG-268, 30 g HA/dose), kindly.