CONSISE workshop on influenza NA and discussion of an international study of the ELLA NA inhibition assay
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1 CONSISE workshop on influenza NA and discussion of an international study of the ELLA NA inhibition assay Maryna Eichelberger Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, US-FDA 1
2 Administrative Information for webinar attendees Webinar attendees are all on mute. Please ask questions or make comments using the chat box. Xianghong Jing will serve as a moderator. She will convey questions/comments at the end of each presentation. Study participants: Please indicate your desire to make a comment or ask a question by posting a message. Study participants who have been notified that they will be unmuted for the discussion: Please keep your microphone on mute until you are ready to make comments. 2
3 Acknowledgements CONSISE organization Maria van Kerkhove Study steering committee John Wood Jackie Katz Min Levine Othmar Engelhardt Karen Laurie Tian Bai Ralf Wagner Catherine Thompson Katja Hoschler Laura Couzens Moderator Xianghong Jing Eichelberger Team Laura Couzens Jin Gao Charu Sharma Giovanna Fantoni Hongquan Wan Lianlian Jiang Sam Mindaye Vaccine Research Center, NIH David Awwad BARDA Funds Yonghong Gao Mario Barro Study participants 15 laboratories (experienced) 20 laboratories (new) 3
4 CONSISE labs participating in ELLA study Public Health England, UK National Institute for Biological Standards and Control (NIBSC), UK University of Bergen, Norway Swedish Inst for Communicable Dis Control, Sweden Erasmus Medical Center, The Netherlands University of Kent, UK Paul-Ehrlich-Institute, Germany Health Canada, Canada GSK, Germany Natl Microbiol Lab, Canada BC CDC, Canada Naval Health, USA Focus Diagnostics, USA Univ of Michigan, USA Baylor Univ, USA CBER, FDA, USA NIAID, NIH, USA Novavax, USA Naval Medical Research Center, USA SJCRH, USA NIH, Portugal Valladolid NIC, Spain Istituto Superiore di Sanita, Italy CDC, USA Institut Pasteur, Algeria National Institute of Virology, India Siriraj Hospital, Mahidol Univerisity, Thailand Labs in blue: experienced Labs in black: new to ELLA Labs underlined: have submitted data Baxter, Austria Cantacuzino Institute, Romania China CDC, China Beijing Inst for Microbiol/Epidemiology, China Univ of Hong Kong Hong Kong Institut Pasteur Cambodia Univ of Melbourne, Australia WHO Influenza Collaborating Centre, Australia 4
5 Study Goals and Design 5
6 Antibodies that inhibit NA activity correlate with protection against disease Murphy et al., 1972; Monto et al., 1973; Clements et al., 1986; Couch et al., 2013 Schulman and Kilbourne 1969; Sandbulte et al. 2007; Marcelin et al., 2010; etc. Determination of NA inhibition (NI) titers Traditional: thio-barbituric acid (TBA) assay Warren, 1959; Aminoff, 1961 Webster and Laver, 1967 Enzyme linked lectin assay (ELLA) Lambre et al., 1990 Cates et al., 2010 Couzens et al., J Virological Methods,
7 Overview of ELLA to determine NI titers 1. Coat plates with fetuin Protein Sugars Galactose Sialic acid 2. Add in standard amount of antigen (H6N1 or H6N2 whole virus) with/without serum dilutions NA Protein Sugars Galactose Sialic acid 3. Add in PNA-HRPO Protein Sugars Galactose::PNA-HRPO 4. Add in substrate (OPD) Protein Sugars Galactose::PNA-HRPO OPD Signal 7
8 Study Goals Determine the reproducibility of ELLA when conducted in different labs Evaluate the benefit of a serum standard Facilitate establishment of ELLA in new labs Identify ELLA reagents/steps to improve performance 8
9 Study plan Perform the ELLA in different laboratories on a single set of 12 samples; measure NA inhibition titers against N1 of A/BR/59/2007 (H1N1) and N2 of A/UR/716/2007 (H3N2) A single SOP used with either OPD or TMB as substrate; data generated with alternate assays run in parallel could be compared 12 samples representing a range of titers were selected for the study; 1 sample was repeated (within assay variability); assay run on 3 different days by the same operator (intra-lab variability) One sample (transgenic bovine plasma, Sanford) was intended to be used as a standard H6N1 and H6N2 reassortant viruses were BPL-inactivated and used in-house to confirm suitability before distribution 9
10 Data Submitted 50% end point titers IC50 (non-linear regression) as secondary measure Assay conditions/reagents Data received to date 14/15 experienced labs 7/20 labs new to ELLA Each lab identified by letter code 10
11 NI titers against A/BR/59/2007 (H1N1) lab S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S 10 S 11 S 12 A B C D E F G H H I J K L M N N O O2 NA NA NA NA Avg
12 NI titers against A/UR/716/2007 (H3N2) lab S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S 10 S 11 S 12 A B C D E F G H H I J K L M N N O O Avg
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