Assessment of memory humoral responses in Hepatitis B vaccine recipients using an in-house B cell Enzyme-Linked Immunospot assay

Abstract

Abstract Introduction: Hepatitis B vaccination has over time provided immense protection to recipients with guidelines endorsing anti-HBsAg titres of ≥ 10IU/ml to correlate with immune protection. hepatitis B surface antibody titres alone may not be a good correlate as it does not measure memory B cells. Protection has been demonstrated in individuals with titres ˂10Ul/ml mediated by memory B cells. Measurement of which isn’t commonly done due to difficulty in detection owing to lack of methods and reagents that allow reliable discrimination of antigen specific cells. This study aimed to establish a B-cell Enzyme-Linked Immunospot (ELISPOT) assay protocol at Virus Research Institute/International Vaccine HIV Vaccine Program (UVRI/IAVI) and evaluate its utility in measuring of hepatitis-B vaccine-induced memory B cells responses among of hepatitis-B virus vaccine recipients. Methods A cultured Enzyme-Linked Immunospot method was followed; 7 Peripheral Blood Mononuclear cells (PBMCs) from vaccinated donors with known and detectable Hepatitis B surface antibody titres and 2 unvaccinated donors with no previous exposure or detectable Hepatitis B surface antibody titres were used to ensure optimal assay conditions. Results Activation of Peripheral Blood Mononuclear cells using Interleukin 2 and R848 cocktail demonstrated highest and superior induction of antibody secreting cells compared to hepatitis-B virus vaccine specific stimulation and the widely used polyclonal activation method using CpG oligodeoxynucleotides (CpG- ODN), Pokeweed mitogen and Staphylococcus aureus Cowans strain cocktail. Use of Interleukin 2 and R848 cocktail possessed an additional advantage of reduction in activation time with an optimal period established after four days of culture. This was however characterized with the production of up to 100,000 antibody secreting cells/1.0x106 Peripheral Blood Mononuclear cells with no Hepatitis B specific antibody secreting cells. Conclusions Our results seemed to suggest that an in-house B cell IgG Enzyme-Linked Immunospot assay may not be the best method to characterize Hepatitis B specific memory B cells. Other Studies to test the use of commercially available B cell epitopes and their validity for use in in-vitro assays could probably help inform efforts to improve the sensitivity of the assay.