Comparative characterization of B cells specific for HBV nucleocapsid and envelope proteins in patients with chronic hepatitis B

•We developed a method for direct ex vivo visualization of HBV-specific B cells. •HBcAg- and HBsAg-specific B cells of patients with CHB differ in phenotype and function. •HBcAg-specific B cells are class-switched memory B cells and secrete antibodies. •HBV-specific B cells express cross-presentation and innate immune genes. •HBV-specific B cell’s functional profiles shadow serological data of patients with CHB. Background & Aims Knowledge about the regulation of anti-HBV humoral immunity during natural HBV infection is limited. We recently utilized dual fluorochrome-conjugated HBsAg to demonstrate, in patients with chronic HBV (CHB) infection, the functional impairment of their HBsAg-specific B cells. However, the features of their HBcAg-specific B cells are unknown. Here we developed a method to directly visualize, select and characterize HBcAg-specific B cells in parallel with HBsAg-specific B cells. Methods Fluorochrome-conjugated HBcAg reagents were synthesized and utilized to directly detect ex vivo HBcAg-specific B cells in 36 patients with CHB. The frequency, phenotype, functional maturation and transcriptomic profile of HBcAg-specific B cells was studied by flow cytometry, in vitro maturation assays and NanoString-based detection of expression of immune genes, which we compared with HBsAg-specific B cells and total B cells. Results HBcAg-specific B cells are present at a higher frequency than HBsAg-specific B cells in patients with CHB and, unlike HBsAg-specific B cells, they mature efficiently into antibody-secreting cells in vitro. Their phenotypic and transcriptomic profiles show that HBcAg-specific B cells are preferentially IgG+ memory B cells. However, despite their phenotypic and functional differences, HBcAg- and HBsAg-specific B cells from patients with CHB share an mRNA expression pattern that differs from global memory B cells and is characterized by high expression of genes indicative of cross-presentation and innate immune activity. Conclusions During chronic HBV infection, a direct relation exists between serological detection of anti-HBs and anti-HBc antibodies, and the quantity and function of their respective specific B cells. However, the transcriptomic analysis performed in HBsAg- and HBcAg-specific B cells suggests additional roles of HBV-specific B cells beyond the production of antibodies. Lay summary Protection of viral infection necessitates the production of antibodies that are generated by specialized cells of the immune system called B cells. During chronic HBV infection, antibodies against the internal part of the virus (core or HBcAg) are detectable while the antibodies directed against the virus envelope (surface or HBsAg) are not present. Here we developed a method that allows us to directly visualize ex vivo the B cells specific for these 2 viral components, highlighting their differences and similarities, and showing how 2 components of the same virus can have different impacts on the function of antiviral B cells. Knowledge about the regulation of anti-HBV humoral immunity during natural HBV infection is limited. We recently utilized dual fluorochrome-conjugated HBsAg to demonstrate, in patients with chronic HBV (CHB) infection, the functional impairment of their HBsAg-specific B cells. However, the features of their HBcAg-specific B cells are unknown. Here we developed a method to directly visualize, select and characterize HBcAg-specific B cells in parallel with HBsAg-specific B cells. Fluorochrome-conjugated HBcAg reagents were synthesized and utilized to directly detect ex vivo HBcAg-specific B cells in 36 patients with CHB. The frequency, phenotype, functional maturation and transcriptomic profile of HBcAg-specific B cells was studied by flow cytometry, in vitro maturation assays and NanoString-based detection of expression of immune genes, which we compared with HBsAg-specific B cells and total B cells. HBcAg-specific B cells are present at a higher frequency than HBsAg-specific B cells in patients with CHB and, unlike HBsAg-specific B cells, they mature efficiently into antibody-secreting cells in vitro. Their phenotypic and transcriptomic profiles show that HBcAg-specific B cells are preferentially IgG+ memory B cells. However, despite their phenotypic and functional differences, HBcAg- and HBsAg-specific B cells from patients with CHB share an mRNA expression pattern that differs from global memory B cells and is characterized by high expression of genes indicative of cross-presentation and innate immune activity. During chronic HBV infection, a direct relation exists between serological detection of anti-HBs and anti-HBc antibodies, and the quantity and function of their respective specific B cells. However, the transcriptomic analysis performed in HBsAg- and HBcAg-specific B cells suggests additional roles of HBV-specific B cells beyond the production of antibodies.