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

Abstract

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.

Interlaboratory Reproducibility of Standardized Hemagglutination Inhibition Assays

“…However, large shifts in the circulating virus reduce the effectiveness if there is a large mismatch between the virus used to create the reference standard and the virus target being evaluated. The work of Carreño et al to develop a reference standard containing anti-stalk antibodies to aid in harmonizing stalk-based immunogenicity assays supports the importance of using a reference standard that is related to the antibody population to be measured ( 11 ).…”

Antibody Responsiveness to Influenza: What Drives It?

“…Since non-HAI antibodies have been identified as additional correlates of protection, it is important to therefore understand their recall dynamics. Efforts are currently underway to incorporate these assays as standard endpoints in influenza studies [138,139].…”

The antibody landscapes against Group 1 and 2 influenza virus hemagglutinin following AS03 and MF59 adjuvanted H5N1 vaccination

“…Antibody directed against HA stalk In order to estimate the relative levels of anti-stalk responses elicited by the 3 vaccine groups, we used a stabilized trimeric Mini H1 HA recombinant stalk molecule 20,21,22 (#4900), produced by Dr. L. Coughlan laboratory), which lacks the entire HA1 head including the HA1 segment that interacts with the stalk. As a negative control we used a pre-fusion stabilized trimeric RSV F protein (DS-Cav1 5K6I, also produced by Dr. L. Coughlan laboratory) 23 .…”