Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza

Abstract

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.

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells

“…Spleen cells were electrofused to the NS0 myeloma cell line using as described previously. 36,37 Hybridoma supernatants were screened for appropriate antigen specificity by ELISA on EGFRvIII and EGFR recombinant proteins. Briefly, half-area 96-well plates (Costar #3690) were coated with 25 mL per well of immunogen at 5 mg/mL in PBS and incubated overnight at 4 C. Microplates were washed three times in PBS and blocked for 30 min with PBS containing 1% bovine serum albumin (BSA; Sigma, catalog #A7030).…”

Masking terminal neo-epitopes of linear peptides through glycosylation favours immune responses towards core epitopes producing parental protein bound antibodies

“…This study further investigated observations from previous work using glycosylated peptide- protein conjugates to raise monoclonal antibodies for quantification of multiple strains of influenza 33 . During these studies, conserved HA peptide epitopes were lactosylated to increase peptide solubility but it was also observed to influence the epitope specificity of the raised antisera.…”

“…Previously, we have demonstrated that the KLH conjugate of a glycosylated HA peptide (a.a. 1–14 HA2) resulted in improved immunogenicity and specificity towards the peptide core sequence 33 . We reasoned these improvements arose from the effect of peptide glycosylation by (1) improvement on peptide solubility, and (2) masking the terminal neo-epitope present on the KLH conjugates.…”

“…Conjugation of saccharides to immunogenic proteins can render them more immunogenic however, it is often observed that these saccharide-protein conjugates produce significant responses against the linkage area 30 as opposed to the saccharide; reinforcing their poor immunogenicity 31 , 32 . The relatively innocuous presence of terminal saccharides was exploited in the development of a pan-HA specific mAb for the quantification of multiple strains of influenza 33 . This study employed the use of a non-disclosed lactosylation technique as one of the measures to increase the solubility of an HA peptide for conjugation purposes, akin to phosphate modifications of HIV lipopeptides 34 .…”

Antigenic Sensitivity of Membrane-Proximal Targeting Chimeric Antigen Receptors can be Fine-Tuned through Hinge Truncation

“…CAR expressing Jurkat cells or primary T cells were generated as indicated elsewhere and assessed for surface expression using an Alexa-Fluor647 labelled murine monoclonal anti-sdAb antibody generated in house. In brief, mice were immunized with sdAb fragments produced in e coli followed by B cell isolation and hybridoma formation as previously reported[32]. Clonal hybridoma supernatants were screened for reactivity against a number of plate bound sdAbs to identify broadly sdAb-reactive monoclonal antibodies.…”