A33 NOVEL E2 GLYCOPROTEIN TETRAMER DETECTS HCV-SPECIFIC MEMORY B CELLS

Hepatitis C virus infection becomes chronic in most cases while a minority (25%) can spontaneously resolve the infection. Antibodies that are specific to the envelope glycoproteins E1 and E2 are generated late during acute infection and yet their role in spontaneous clearance remains debated. Some reports described cases of viral clearance without seroconversion. Other studies demonstrated an increased rate of spontaneous resolution in individuals who developed an early HCV-specific antibody response. Investigation of the humoral responses during acute HCV infection requires identification of HCV-specific B cells. We hypothesized that expression of a biotinylated form of the ectodomain of E2 glycoprotein and fluorescent tetramer generation would enable detection of HCV-specific B cells from patient peripheral blood samples. We have developed an expression vector enabling production and purification of biotinylated HCV E2 ectodomain. Fluorescent tetramers were generated by incubation with fluorescent streptavidin and they were used together with phenotypic B cells surface markers in flow cytometry experiments (FACS). Longitudinal PBMC samples from individuals who became chronically infected with HCV were used to detect HCV-specific B cells. The new B cell tetramer was also used to FACS sort HCV-specific class-switched memory B cells to perform B cell receptor (BCR) deep sequencing. Our newly developed tetramer enabled us to detect HCV E2 specific memory B cells in most samples of HCV chronic infection (28/31). However, in a longitudinal study, we could detect HCV E2 specific B cells in only half of acute infection samples analysed (3/7), suggesting that in some subject the development of the humoral response might be delayed. We successfully isolated HCV E2 specific class-switched memory B cells from two samples and performed BCR deep sequencing. The BCR repertoire of both samples was focussed and had accumulated mutations, suggesting amplification of particular clonotypes and affinity maturation process. Finally, our results suggest that our newly developed B cell tetramer will be very useful to study the development of the humoral response to HCV infection. We showed that most infected individuals developed HCV-specific B cell population, but in some cases, HCV-specific B cells could only be detected during the chronic phase of the infection. Future studies examining subjects that spontaneously resolved the infection will enable characterization of the protective immune response and define the role of the humoral response in spontaneous clearance of HCV infection. CIHR

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