RG viruses were generated by reverse genetics as described in (Shcherbik et al
RG viruses were generated by reverse genetics as described in (Shcherbik et al., 2015). Influenza, Neuraminidase, Monoclonal antibody, Live vaccine, Classical reassortment 1.?Introduction Influenza A virus causes substantial human disease and serious economic burdens worldwide (Gordon and Reingold, 2018; WHO, 2018). Vaccination is the most effective way to prevent influenza infections and reduce the disease severity (Houser and Subbarao, 2015; Osterholm et al., 2012). There are two major types of influenza vaccines licensed for human use: inactivated influenza vaccine, and live attenuated influenza vaccine (LAIV), which is administered intranasally. LAIVs have proven to be an Abscisic Acid effective public health tool in Russia (Aleksandrova, 1977b; Rudenko et al., 1996), USA (Ambrose et al., 2008; Bandell et al., 2011) and Europe (Bandell and Simoes, 2015). LAIV seed viruses are reassortants containing surface antigens (hemagglutinin, HA and neuraminidase, NA) seasonal influenza viruses on the backbone (include M, NA, NP, PA, PB1 and PB2 gene segments) of cold adapted master donor virus (MDV). MDV provides temperature Abscisic Acid sensitive, cold adapted and attenuated phenotype through cooperative multi-gene mutations, typically in PA, PB1 and PB2 polymerase gene segments. These genetic/phenotypic characteristics enable the LAIV reassortants to replicate efficiently at lower temperatures at the upper respiratory tract, restrict replication at the lower respiratory tract and attenuate the virus (Maassab and Bryant, 1999; Murphy and Coelingh, 2002). LAIVs confer protection by inducing neutralizing anti-HA antibodies (Belshe et al., 2000; Cox et al., 2004; Gerhard, Abscisic Acid 2001) and mucosal cellular responses. LAIVs also provides heterosubtypic protection through cross-reactive T-cell responses to conserved epitopes (Epstein and Price, 2010; Haaheim and Katz, 2011; He et al., 2006; Hoft et al., 2011). LAIVs were developed and have been in use in Russia since 1960, they were licensed for use in North America in 2003 (Flumist), Europe has licensed LAIV (Fluenz) in 2013. Through cooperation with WHO, the production and use of LAIVs on backbone of A/Leningrad/134/17/57 (H2N2) master donor virus (Len/17-MDV) has been expanded internationally (Neuzil et al., 2012; Rudenko et al., 2011, 2016). For the preparation of seasonal LAIV seed viruses from Len/17-MDV, WHO Expert Committee on Biological Standardization recommends the conventional reassortment procedure prepared in eggs (WHO, 2013). Rapid and efficient selection of reassortants with 6:2 genome compositions is essential for the tight schedule of vaccine production. The protocol for generating LAIVs based on Len/17-MDV was originally developed by Institute of Experimental Medicine, Russia (Aleksandrova, 1977a; Wareing et al., 2002). The protocol includes 2 selective passages and 1 biological cloning by limiting dilution step performed under selective conditions, in the presence of anti-serum against Len/17-MDV and at low temperature (25 C) to allow the correct 6:2 reassortants to dominate the pool. However, even under such robust selective pressure, variability of the gene segments donated by the cold-adapted donor in the vaccine reassortants was often observed. It was reported that less than 15% of LAIV reassortants derived from post-year 2000 viruses possessed the desired NA (Kiseleva et al., 2014). It has been reported that specific antibodies against HA and NA enhance incorporation of the HA and NA segments from seasonal viruses into vaccine reassortants of viruses with a A/Puerto Rico/8/1934 (H1N1) Abscisic Acid backbone (Stohr GluN1 et al., 2012). To determine if addition of mAb against Len/17-MDV NA increase the efficiency of obtaining desired LAIV reassortants, we developed a new Abscisic Acid anti-neuraminidase mAb using purified Len/17-MDV NA. We found the mAb efficiently and specifically inhibit viruses containing Len/17-MDV NA (N2 subtype) both and and decreases the amount of N2 Len/17-MDV gene segment in the reassortment pool enabling the reassortants with desired NA to prevail in selection steps for production of 6:2 reassortant viruses. 2.?Materials and methods 2.1. Viruses, cells, antibodies A/Leningrad/134/17/57 (H2N2) (Len/17-MDV) was provided by BioDiem (Australia). LAIV viruses A/Texas/50/2012(H3N2)-CDC-LV4A (LV4A), A/South Africa/3646/2013(H1N1pdm09)-CDC-LV14A (LV14A), A/Hong Kong/4801/2014(H3N2) CDC-LV15A (LV15A) were generated using classical reassortment with Len/17-MDV in specific pathogen free (SPF) eggs (Charles River Laboratories Inc., USA). The viruses used.