Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies
Juan Manuel Carreño,
Jacqueline U. McDonald,
Tara Hurst,
Peter Rigsby,
Eleanor Atkinson,
Lethia Charles,
Raffael Nachbagauer,
Mohammad Amin Behzadi,
Shirin Strohmeier,
Lynda Coughlan,
Teresa Aydillo,
Boerries Brandenburg,
Adolfo García-Sastre,
Krisztian Kaszas,
Min Z. Levine,
Alessandro Manenti,
Adrian B. McDermott,
Emanuele Montomoli,
Leacky Muchene,
Sandeep R. Narpala,
Ranawaka A. P. M. Perera,
Nadine C. Salisch,
Sophie A. Valkenburg,
Fan Zhou,
Othmar G. Engelhardt,
Florian Krammer
Affiliations
Juan Manuel Carreño
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Jacqueline U. McDonald
Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Tara Hurst
Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Peter Rigsby
Division of Analytical and Biological Sciences, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Eleanor Atkinson
Division of Analytical and Biological Sciences, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Lethia Charles
Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Raffael Nachbagauer
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Mohammad Amin Behzadi
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Shirin Strohmeier
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Lynda Coughlan
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Teresa Aydillo
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Boerries Brandenburg
Janssen Vaccines & Prevention BV, 2333 CP Leiden, The Netherlands
Adolfo García-Sastre
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
Krisztian Kaszas
Janssen Vaccines & Prevention BV, 2333 CP Leiden, The Netherlands
Min Z. Levine
Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA
Alessandro Manenti
VisMederi Research srl, 53100 Siena, Italy
Adrian B. McDermott
Vaccine Immunology Program (VIP), Vaccine Research Center (VRC), National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA
Emanuele Montomoli
Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy
Leacky Muchene
Janssen Vaccines & Prevention BV, 2333 CP Leiden, The Netherlands
Sandeep R. Narpala
Vaccine Immunology Program (VIP), Vaccine Research Center (VRC), National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA
Ranawaka A. P. M. Perera
School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Nadine C. Salisch
Janssen Vaccines & Prevention BV, 2333 CP Leiden, The Netherlands
Sophie A. Valkenburg
School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Fan Zhou
Influenza Center, Department of Clinical Science, University of Bergen, 5021 Bergen, Norway
Othmar G. Engelhardt
Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar EN6 3QG, UK
Florian Krammer
Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA
The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.
