Journal of Virological Methods 237 (2016) 154–158 Contents lists available at ScienceDirect Journal of Virological Methods j o ur nal ho me pag e: www.elsevier.com/locate/jviromet Titration of individual strains in trivalent live-attenuated influenza vaccine without neutralization Naraporn Sirinonthanawech a , Somchaiya Surichan b , Aphinya Namsai b , Pilaipan Puthavathana c , Prasert Auewarakul a,d , Alita Kongchanagul a,∗ a Institute of Molecular Biosciences (MB), Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand b The Government Pharmaceutical Organization (GPO), 75/1 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand c Faculty of Medical Technology, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand d Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok, 10700, Thailand Article history: Received 7 June 2016 Received in revised form 1 September 2016 Accepted 1 September 2016 Available online 3 September 2016 Keywords: Live-attenuated influenza vaccine Influenza virus Trivalent vaccine titration a b s t r a c t Formulation and quality control of trivalent live-attenuated influenza vaccine requires titration of infec- tivity of individual strains in the trivalent mix. This is usually performed by selective neutralization of two of the three strains and titration of the un-neutralized strain in cell culture or embryonated eggs. This procedure requires standard sera with high neutralizing titer against each of the three strains. Obtaining standard sera, which can specifically neutralize only the corresponding strain of influenza viruses and is able to completely neutralize high concentration of virus in the vaccine samples, can be a problem for many vaccine manufacturers as vaccine stocks usually have very high viral titers and complete neutral- ization may not be obtained. Here an alternative approach for titration of individual strain in trivalent vaccine without the selective neutralization is presented. This was done by detecting individual strains with specific antibodies in an end-point titration of a trivalent vaccine in cell culture. Similar titers were observed in monovalent and trivalent vaccines for influenza A H3N2 and influenza B strains, whereas the influenza A H1N1 strain did not grow well in cell culture. Viral interference among the vaccine strains was not observed. Therefore, providing that vaccine strains grow well in cell culture, this assay can reliably determine the potency of individual strains in trivalent live-attenuated influenza vaccines. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Live-attenuated influenza vaccine (LAIV) is being widely used in many countries (Wareing and Tannock, 2001; Jin and Subbarao, 2015). The manufacturing process is less complicated and the yield per embryonated egg is much higher than inactivated vaccines (Rudenko and Isakova-Sivak, 2015). This is an attractive option for vaccine production in a pandemic, when the number of embry- onated eggs is the main limiting factor for total production capacity (Luke and Subbarao, 2006; Rudenko and Isakova-Sivak, 2015). The World Health Organization has been supporting establish- ment of LAIV production capacity in developing countries using the A/Leningrad/134/17/57 master donor strain as part of the pandemic preparedness plan (Friede, 2011; Rudenko et al., 2011; Surichan et al., 2011). ∗ Corresponding author. E-mail address: alita.kon@mahidol.ac.th (A. Kongchanagul). The production of LAIV involves growth of individual vaccine strains in embryonated eggs, partial purification of the viruses from allantoic fluid, titration of monovalent concentrated bulks and for- mulation into trivalent vaccine by mixing the separate strains to obtain the desired amount of infectious virus (WHO, 2013). The trivalent vaccine preparation needs to be checked for the infectiv- ity of each strain. The titration of individual strains in the trivalent vaccine preparation is also needed for evaluating stability of the vaccine preparation after specified storage conditions. Titration of individual strains in trivalent LAIV is usually per- formed by selective neutralization of two of the three strains by standard sera and end-point titration of the un-neutralized strain in embryonated eggs as EID 50 (50% egg infectious dose) or alter- natively in cell culture as TCID 50 (50% tissue culture infectious dose). This procedure requires specific sera with very high neutral- izing titer against each of the vaccine strains (Yeolekar and Dhere, 2012). The standard sera are produced by immunizing specific pathogen free animals with purified hemagglutinin of the respec- tive vaccine strains (WHO, 2013). This can be time-consuming and presents an important obstacle for vaccine production in particu- http://dx.doi.org/10.1016/j.jviromet.2016.09.001 0166-0934/© 2016 Elsevier B.V. All rights reserved.