Use of ELISpot assay to study HBs-specific B cell responses in vaccinated and HBV infected humans

Figures & data

Fig. 1 HBs-specific B cells in health volunteers.

a Representative B-cell ELISpot readouts from healthy volunteers HC9 and HC21. 100,000 cells were added in HBsAg-coated wells (up panel) and negative wells (middle panel) and 5000 cells were added in IgG coated wells (bottom panel). b The frequency of HBs-specific memory B cells was significantly reduced in groups with lower level of serum HBsAb (<200 mIU/ml), compared to the groups with higher serum HBsAb ( > 200 mIU/ml). c A strong correlation between the serological HBsAb and the number of memory B cell was detected in HCs. SFC, spot-forming cells. Each dot represents one individual

Fig. 2 HBs-specific B cells in healthy volunteers post immunization.

a Serum HBsAb titers increased persistently in all immunized healthy subjects at day 7 and day 28 post HBV vaccine boost. b Representative B-cell ELISpot readouts (HC18), who had already finished HBV vaccine immunization 2 years ago, at day 0 (top), day 7 (middle) and day 28 (bottom) after HBV vaccine boost were shown. c HBs-specific B-cell responses were measured either at day 7 or day 28 after boost. *P < 0.05

Fig. 3 HBs-specific B cells in chronic hepatitis B (CHB) patients.

a RepresentativeELISpot data from two CHB patients P27 and P23 were shown. Each condition was performed in duplicates. b Patients with lower level of serum HBsAb (<1 mIU/ml) had relatively fewer HBsAg-specific B cells, compared to those patients with higher level of serum HBsAb (>1 mIU/mL). c The numbers of HBs-specific B cells among 10⁶ PBMC population were shown in each individual from HC group and CHB patient group

Clinical characteristics of CHB patients and HCs in the study

Group	IT	IA	IC	HC
No. of Cases	13	17	21	21
Gender (M/F)	10/3	11/6	17/4	8/13
Age (years)	31(27–33)	28(26–34)	36(30–39)	27(25–34)
HBsAg	+	+	+	−
HBeAg	+	+	−	−
ALT (U/L)	32.9	104	27.1	18.9
	(23.95–45.5)	(73.5–150)	(19.7–37.1)	(9.475–33.8)
HBV DNA	7.528	6.812	ND	ND
(log10 IU/ml)	(7.33–7.826)	(5.318–7.61)

HC healthy controls, IA immune reactive HBeAg-positive phase, IC Inactive HBV carrier state, IT Immune tolerant phase, ND not detectable, ALT alanine transaminase. Data are shown as median and range

Fig. 4 HBs-specific B cells in CHB patients.

a The positive rates of HBs-specific B cells in immune tolerant (IT), immune reactive HBeAg-positive (IA), and inactive HBV carrier (IC) phases were expressed as a percentage. b The frequencies of HBsAb-SFCs among three CHB phases were displayed. **p < 0.01

Fig. 5 HBV Vaccine-elicited memory B-cell response in CHB patients.

a Representative B-cell ELISpot readouts (patient VP6) at days 0 (left), 7 (middle) and 28 (right) after booster. b Serum HBsAb titers and (c) HBs-specific B cells were quantitatively measured from 12 CHB patients at days 0, 7, and 28 after receiving the HBV vaccination

Fig. 6 HBs-Specific memory B cell response with PEG-IFN treatment.

a Therapeutic regimens of four CHB patients. Patient 1, 2, and 3 had combination of PEG-IFN with nucleoside analogs while patient 4 had PEG-IFN treatment alone. b Serum HBsAg levels, HBs-specific B cells and serum HBsAb titers before (white column) and after (black column) PEG-IFN treatment of these 4 patients were displayed. ETV entecavir, ADV adefovir dipivoxil, LAM lamivudine, TDF tenofovir