Establishment of H5-specific ELISpot and B cell flow cytometry to investigate memory B cell responses induced by avian influenza A(H5N8) vaccination

Abstract

Highly pathogenic avian influenza A(H5N1) virus has resulted in several outbreaks in domestic and wild birds globally, creating the need for a vaccine to prevent human transmission. Zoonotic influenza A(H5N8) vaccine by Seqirus was approved in 2023 in order to protect against H5 subtype influenza A viruses, including the highly pathogenic clade 2.3.4.4b A(H5N1) avian influenza virus infection. The B cell responses this vaccine induces have not been extensively investigated. Thus, the aim of this study was to develop hemagglutinin H5 specific ELISpot and memory B cell flow cytometry to investigate B cell responses induced by the H5N8 vaccine.

Stored PBMCs from H5N8-vaccinated and non-H5N8-vaccinated individuals were used for setting up the methods. For ELISpot, PBMCs were incubated with R848 and IL-2 to activate B cells. Subsequently, antibody secreting cells specific for recombinant HA-proteins of H5 and H1 subtypes, and H5N1 vaccine (A/Indonesia/05/2005 Vaccine), as well as for SARS-CoV-2 S1 and IgG, were detected on coated ELISpot plates. The detection of the antibody-secreting cells was optimized by testing a two-step detection method using biotin and streptavidin-alkaline phosphatase, after which it was compared to a one-step detection method using alkaline phosphatase. For memory B cell flow cytometry, PBMCs were labelled with anti-CD3, anti-CD19, and anti-IgD. The H5 HA-specific cells were detected with additional dual-labelling using lighting link AF647, and PE-probed recombinant HA1-subunit of H5. To achieve optimal detection, the optimization included testing T-cell depletion and different amounts of dual-labelled H5 HA.

As a result, we were able to develop functional H5 HA-specific ELISpot and memory B cell flow cytometry. In ELISpot, SARS-CoV-2 S1-specific antibody-secreting cells were detected in all study participants, and the number of H5 HA- and H5N1 vaccine-specific responses were increased in H5N8 vaccinees. In the memory B cell flow cytometry, the number of H5 HA-specific memory B cells was four times higher in the samples from H5N8 vaccinees than from the non-H5N8-vaccinees. Due to the limited sample size, these results should be considered preliminary, and further studies with larger sample cohorts are required to properly assess the quantitative changes of these responses.