Abstract
Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.
Highlights
A successful and durable adaptive immune response comprises both expansion of short-lived effectors and development of long-lived memory cells
Using an affinity capture coating approach leveraging the genetically encoded hexahistidine (6XHis) affinity tag commonly expressed by recombinant proteins, we introduce a universal platform for antigen absorption that improved the sensitivity of B-cell ImmunoSpot assays
Using murine B-cell hybridomas and in vitro differentiated human memory B cells as model antibody-secreting cells (ASC), we demonstrate the utility of the affinity capture approach across a variety of recombinant proteins: seasonal influenza (H1N1 and H3N2), latent herpesviruses (EBV and human cytomegalovirus (HCMV)) and most notably the pandemic SARS-CoV-2 virus
Summary
A successful and durable adaptive immune response comprises both expansion of short-lived effectors and development of long-lived memory cells. With the exception of following acute antigen exposure, in which elevated frequencies of short-lived ASC can be found in circulation and transiently alter serum antibody reactivity [3,4,5], long-lived plasma cells (LLPC) residing in the bone marrow and secondary lymphoid tissues are responsible under steady state for the composition and specificity of serum antibody (reviewed by [6]). Short-lived ASC are generated during T-cell-dependent immune responses, yet only a subset of these ASC acquire the appropriate gene expression program and successfully occupy the specialized niche required for long-term survival and maintenance of sustained antibody production (reviewed by [9,10,11])
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