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Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015–2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains. [ABSTRACT FROM AUTHOR]

Vaccination is the most effective prophylactic method for preventing influenza. Quantification of influenza vaccine antigens is critically important before the vaccine is used for human immunization. Currently the vaccine antigen quantification relies on hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the sialic acid binding property of influenza vaccine antigens. Specifically, we chemically synthesized human and avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two sialyllactose-derivatives was reduced and conjugated to mouse serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived hemagglutinins, respectively, forming the basis of a new, and robust assay for hemagglutinin quantification. Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple influenza vaccines. [ABSTRACT FROM AUTHOR]