Abstract
Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA+ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.
Highlights
The surface glycoprotein hemagglutinin (HA) plays a critical role in influenza virus infection, by anchoring viruses to surface sialic-acid residues on host cells and by mediating the subsequent fusion of viral and host cell membranes
To identify B-cells engaged into BCR-specific interactions with influenza HA we first tried to stain PBMCs with monoclonal antibodies against the B-cell marker CD20 and the B-cell memory marker CD27 mixed with a recombinant H1 bait, or with human serum albumin (HSA), both conjugated with the Alexa-488 fluorochrome (A488)
When stained with rHA, PBMCs gated on live singlets (Fig. 1A) showed a high and diffuse fluorescent signal on both B and non B-cells, while staining with HSA-A488 only gave background signal (Fig. 1B)
Summary
The surface glycoprotein hemagglutinin (HA) plays a critical role in influenza virus infection, by anchoring viruses to surface sialic-acid residues on host cells and by mediating the subsequent fusion of viral and host cell membranes. Antibodies blocking these interactions are the only widely recognized correlate of protection from infection. Influenza HA is highly susceptible to mutations and drifted variants capable to escape pre-existing neutralizing antibodies emerge continuously. For this reason influenza vaccines must be reformulated yearly. To what extent, pre-existing memory B-cells (MBCs) play a role in preventing infection by new influenza variants is poorly understood [4,5]
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