Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

Carreño, Juan Manuel and McDonald, Jacqueline U. and Hurst, Tara and Rigsby, Peter and Atkinson, Eleanor and Charles, Lethia and Nachbagauer, Raffael and Behzadi, Mohammad Amin and Strohmeier, Shirin and Coughlan, Lynda and Aydillo, Teresa and Brandenburg, Boerries and García-Sastre, Adolfo and Kaszas, Krisztian and Levine, Min Z. and Manenti, Alessandro and McDermott, Adrian B. and Montomoli, Emanuele and Muchene, Leacky and Narpala, Sandeep R. and Perera, Ranawaka A. P. M. and Salisch, Nadine C. and Valkenburg, Sophie A. and Zhou, Fan and Engelhardt, Othmar G. and Krammer, Florian (2020) Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies. Vaccines, 8 (4). e666. ISSN 2076-393X

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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.

Item Type: Article
Additional Information: ** From MDPI via Jisc Publications Router ** History: accepted 04-11-2020; pub-electronic 09-11-2020. ** Licence for this article:
Identification Number:
4 November 2020Accepted
9 November 2020Published
Uncontrolled Keywords: influenza vaccine, serology, hemagglutinin, stalk, standardization
Subjects: CAH01 - medicine and dentistry > CAH01-01 - medicine and dentistry > CAH01-01-01 - medical sciences (non-specific)
Divisions: Faculty of Health, Education and Life Sciences > School of Health Sciences
SWORD Depositor: JISC PubRouter
Depositing User: JISC PubRouter
Date Deposited: 18 Dec 2020 12:00
Last Modified: 12 Jan 2022 11:13

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