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

Leacky Muchene,Shirin Strohmeier,Sandeep Narpala ,Fan Zhou,Eleanor Atkinson,Tara Hurst,Peter Rigsby,Min Z. Levine,Emanuele Montomoli,Sophie A. Valkenburg,Raffael Nachbagauer,Mohammad Amin Behzadi,Juan Manuel Carreño,Lynda Coughlan,Alessandro Manenti,Lethia Charles,Ranawaka A.p.m. Perera ,Adrian B. McDermott,Boerries Brandenburg,Jacqueline U. McDonald,Adolfo Garcı́a-Sastre ,Krisztián Kaszás ,Florian Krammer,Nadine C. Salisch,Teresa Aydillo

doi:10.3390/vaccines8040666

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.

Highlights

As defined in the strategic plan from the National Institute of Allergy and Infectious Diseases [1], some of the key points to achieve the development of effective Universal Influenza Vaccines (UIV)include: the characterization of the immune responses elicited during influenza virus infection and vaccination; establishment of novel non-hemagglutination inhibition (HAI) correlates of protection; rational design of antigens with a wider breadth of protection; and implementation of these candidates in phase I-II clinical studies
Several studies have demonstrated that some individuals possess higher levels of antibodies directed to the HA stalk of influenza viruses, and that these antibodies increase over time due to multiple exposures with influenza virus strains that are antigenically related [37,38]
Humans are naïve to the exotic avian H6 head domain, an undetectable amount of anti-head antibodies is present in human serum samples [39]

Summary

Introduction

As defined in the strategic plan from the National Institute of Allergy and Infectious Diseases [1], some of the key points to achieve the development of effective Universal Influenza Vaccines (UIV). Include: the characterization of the immune responses elicited during influenza virus infection and vaccination; establishment of novel non-hemagglutination inhibition (HAI) correlates of protection; rational design of antigens with a wider breadth of protection; and implementation of these candidates in phase I-II clinical studies. Many current efforts towards the development of these novel types of vaccines rely on the induction of effective long-term antibody responses against conserved regions of the influenza virus glycoproteins [2]. The stalk domain of the hemagglutinin (HA) has been identified as a suitable target for universal influenza virus vaccines due to its unique properties. Several vaccine candidates targeting this domain are in late pre-clinical, or early clinical stages of development [2,18,19,26,27]

Methods

Results

Conclusion