Abstract
Determination of the frequency of specific B lymphocytes has important implications for investigation of the immune response to different antigens and pathogens. Unresponsiveness to some viruses and antigens, such as hepatitis B virus (HBV) and its surface antigen (HBsAg), has been attributed to lack or insufficient production of the specific B-cell repertoire. In this study, peripheral blood B lymphocytes of 45 adult normal individuals vaccinated with recombinant hepatitis B vaccine were transformed with Epstein–Barr virus (EBV) and cultured at different dilutions on human fetal fibroblasts as a feeder layer. The vaccinees were classified into good, poor and non-responder groups. Following 2 to 3 weeks of incubation, culture supernatants were collected from wells containing transformed and proliferating B lymphocytes. The supernatants were subsequently screened for the presence of total immunoglobulin and antibody to HBsAg (anti-HBs) by enzyme-linked immunosorbent assay (ELISA). Accordingly, positive and negative wells were enumerated in each plate and the frequency of B lymphocytes producing anti-HBs antibody was estimated based on the Poisson statistical analysis. The total number of CD19+ B lymphocytes were counted in the peripheral blood of all subjects by flow cytometry. Our results demonstrated a similar precursor frequency of specific B lymphocytes in all subject groups before vaccine administration (< 2 × 10−5). Following vaccination, however, a significant increase in the number of specific B lymphocytes was observed in good-responder (1·5 × 10−4) and to a lesser extent poor-responder (3·5 × 10−5) individuals, but not in non-responders. These findings suggest a defect in either the primary B-cell repertoire or helper T-cell function in non-responder individuals.
Introduction
Hepatitis B virus (HBV) infection is a worldwide health problem with an increased incidence in developing countries.1 Exposure of healthy adult individuals to HBV results in a protective antibody response in 90–95% of subjects, associated with either an asymptomatic or an acute clinical course.2 Vaccination with the major surface protein of HBV – hepatitis B surface antigen (HBsAg) – has also been found to induce a protective antibody response in a similar proportion of the normal adult population and also in neonates and children.2,3 However, 5–10% of the vaccinees display an inadequate antibody response following primary vaccination with triple doses of either plasma-derived or recombinant hepatitis B vaccine. These individuals remain at risk of infection with HBV.4
Different mechanisms may contribute to the lack of a specific antibody response to HBsAg. Some findings suggest a lack or insufficient production of HBsAg-specific precursor B cells in non-responder subjects.5–8 These results, however, have been derived mainly from in vitro lymphocyte stimulation studies. Direct determination of the frequency of specific B lymphocytes have important implications for investigation of the immune response to different antigens and pathogens.9
Human precursor B cells can be transformed by Epstein–Barr virus (EBV) in vitro to lymphoblastoid cell lines without losing their capability to produce immunoglobulin. EBV transformation in combination with limiting dilution assay (LDA) can be used to evaluate the frequency of precursor B cells producing antigen-specific antibodies.10,11
In the present study, this methodology was used for the first time to estimate the frequency of HBsAg-specific B lymphocytes in responder and non-responder normal individuals vaccinated with recombinant hepatitis B vaccine.
Materials and methods
Subjects and vaccination schedule
A total of 45 healthy adult individuals (34 males, 11 females, 17–61 years of age), who were negative for HBsAg and antibody to hepatitis B core antigen (anti-HBc) and antibody to hepatitis B surface antigen (anti-HBs), were vaccinated with triple 20-µg doses of recombinant hepatitis B vaccine (Heberbiovac, S.A. Havana, Cuba), as previously described.3,12 Two weeks after completion of vaccination, the serum level of anti-HBs antibody and the frequency of HBsAg-specific B lymphocytes were determined. Based on the titre of anti-HBs antibody, the vaccinees were arbitrarily classified into three groups: good responders (n = 34) (anti-HBs antibody > 100 IU/l), poor responders (n = 5) (anti-HBs antibody 10–100 IU/l) and non-responders (n = 6) (anti-HBs antibody < 10 IU/l).
EBV transformation and limiting dilution of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized peripheral venous blood by Ficoll–Paque (Sigma, St Louis, MO) centrifugation and transformed with EBV, as described previously.13,14 Briefly, the cells were resuspended in EBV filtrate produced by EBV-infected B95.8 marmoset cells (NCBI C-110; National Cell Bank of Iran, Tehran, Iran). After 1 hr of incubation at 37°, with periodic agitation, the cells were washed with RPMI-1640 (Sigma) and resuspended in the same medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics, including penicillin (100 U/ml) and streptomycin (100 µg/ml). Treated cells were prepared in four different concentrations (4 × 104, 8 × 104, 1·6 × 105 and 3·2 × 105), and a sample representing each concentration was added to each of 60 wells of a 96-well microculture plate (Griner, Nötingen, Germany), on human fetal foreskin fibroblasts (HFFF-PI6) (NCBI C-170; National Cell Bank of Iran). Memory T lymphocytes of individuals seropositive for EBV, which usually attack the EBV-infected B lymphocytes, were inhibited by addition of 1 µg/ml of cyclosporine A (Sandoz, Basel, Switzerland) to the culture medium. Following 2–3 weeks of incubation, culture supernatants were collected from wells containing transformed and proliferating B lymphocytes. The supernatants were subsequently screened for the presence of total immunoglobulin and anti-HBs antibody.
Detection of HBV markers
Anti-HBs antibody, anti-HBc antibody and HBsAg were detected by enzyme-linked immunosorbent assay (ELISA), using commercial kits (Behring, Marburg, Germany). Anti-HBs antibody was quantified in serum and culture supernatant by comparison to a reference curve, which was produced using appropriate dilutions of a positive sample with known concentrations of the antibody (expressed as IU/l) (Behring).
Detection of human immunoglobulin
An indirect ELISA was used to detect total immunoglobulin in the culture supernatant. Briefly, microtitre ELISA plates (Griner) were sensitized with 5 µg/ml of goat anti-human immunoglobulin (polyvalent) (Sigma). Human immunoglobulin was detected by using rabbit anti-human immunoglobulin F(ab′)2-conjugated horseradish peroxidase (HRP) (Sigma). Following addition of the substrate tetramethylbenzidine (TMB; Behring), the absorbance values of the samples were recorded by a multiscan ELISA reader (Organon Teknika, Boxtel, the Netherlands).
Enumeration of B lymphocytes in PBMC
PBMC (0·5–1 × 106) were washed twice with cold phosphate-buffered saline (PBS) (pH = 7·4, 0·15 m) and incubated with fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal anti-human CD19 (F 07689, DAKO, Glostrup, Denmark) and an isotype-matched negative control, in the dark at 4° for 30 min. After incubation, the cells were washed twice with 2% PBS supplemented with bovine serum albumin (BSA) (Sigma). The cells were then fixed in 1% paraformaldehyde solution before analysis using a fluorescence-activated cell sorter (FACScan; Becton-Dickinson, CA).
Determination of the efficiency of EBV transformation in PBMC
PBMC were infected with EBV (as described above) and seeded, at 125, 250, 500 and 1000 cells/well, into 96-well tissue culture plates (Griner) in the presence of feeder cells (HFFFP-I6). Each cell density was distributed into 60 wells. The frequency of transformed B lymphocytes in PBMC was evaluated based on the Poisson statistical analysis and percentage of B lymphocytes in PBMC.
Determination of the frequency of HBsAg-specific B lymphocytes in PBMC
The frequency of HBsAg-specific B lymphocytes in the PBMC of all vaccinees was determined before and after hepatitis B vaccination. Positive and negative wells were identified after screening all wells of each dilution for anti-HBs antibody, and the frequency of B lymphocytes producing anti-HBs antibody was estimated based on the Poisson statistical analysis. This estimation was calculated taking into account the percentage of B lymphocytes in PBMC and the efficiency of transformation with EBV.
Statistical analysis
The frequency of HBsAg-specific B lymphocytes was calculated from LDA by Poisson analysis.15 Comparison of variables was analysed using the t-test, and correlation between the frequency of HBsAg-specific B lymphocytes and the titre of anti-HBs antibody in the responder group was assessed by regression analysis using the spss statistical package. P-values of less than 0·05 were considered significant.
Results
Measurement of anti-HBs antibody in serum
The level of anti-HBs antibody was measured in the serum of all subjects before and after vaccination. A detectable level of anti-HBs antibody was not identified before vaccination in any of the individuals. The results obtained after completion of vaccination are shown in Fig. 1. The mean titre of anti-HBs antibody was found to be 23 438, 67 and 3·3 IU/l in good, poor and non-responder groups, respectively.
Figure 1.
Serum levels of antibody to hepatitis B surface antigen (anti-HBs antibody) in responder and non-responder vaccinees.
Frequency of anti-HBs antibody-producing B cells in PBMC
EBV-infected PBMC from all responder and non-responder individuals were seeded at 40 000, 80 000, 160 000 and 320 000 cells/well (60 wells for each dilution), and the number of anti-HBs antibody-positive wells were subsequently counted (Table 1). Comparison of the results obtained for each dilution in responder and non-responder groups revealed highly significant differences between all the responder groups, particularly between good responder and non-responder groups and between good responder and poor-responder groups (Table 2). The proportion of negative wells of the total number of wells seeded (60 wells) was calculated for each cell dilution and plotted as negative fraction against cell dilution rate, to estimate the cell frequency according to the Poisson equation. Negative wells are defined as containing growing cells with culture supernatants positive for total human immunoglobulin, but negative for anti-HBs antibody. The absorbance values obtained by ELISA for total human immunoglobulin were greater than 1·5 in all wells tested, equivalent to a concentration of more than 500 ng/ml, as extrapolated from a standard curve constructed from known concentrations of affinity-purified human IgM (data not shown). Representative curves obtained for good and poor responders are shown in Fig. 2. These curves were plotted for all vaccinees and the primary frequency of specific B cells was deduced based on the Poisson statistical analysis. To determine the number of HBsAg-specific B lymphocytes, the percentage of B cells in PBMC and the efficiency of EBV transformation needed to be calculated and taken into consideration. The efficiency of B-cell transformation by EBV was found to be 3% (data not shown). The mean percentage of CD19+ B cells was similar in all responder and non-responder groups (14–16%) (Table 3). Accordingly, the mean frequencies of specific B cells after vaccination were found to be 1/6654, 1/28 505 and < 1/49 115 for good, poor and non-responder groups, respectively (Table 4). A significant difference was observed for good responders as compared to poor- and non-responder groups (P < 0·02 and P < 0·0001, respectively). The frequency of specific B cells before vaccination was similar for all vaccinated groups (< 1/49 115).
Table 1.
Screening of wells positive for antibody to hepatitis B surface antigen (anti-HBs antibody) in responder and non-responder groups
| No. of anti-HBs antibody-positive wells at each dilution | ||||
|---|---|---|---|---|
| Subjects | 320 000* | 160 000 | 80 000 | 40 000 |
| Good responders (A) | 2–52 | 0–45 | 2–30 | 1–11 |
| (n = 34) | (21 ± 16) | (14 ± 12) | (9 ± 8) | (4 ± 3) |
| Poor responders (B) | 1–3 | 0–4 | 0–2 | 0–2 |
| (n = 5) | (2 ± 1) | (2 ± 3) | (1 ± 0·9) | (0·7 ± 0·9) |
| Non-responders (C) | 0–1 | 0 | 0 | 0 |
| (n = 6) | (0·2 ± 0·3) | |||
Epstein–Barr virus (EBV)-infected peripheral blood mononuclear cells (PBMC) were prepared at dilutions corresponding to 40 000, 80 000, 160 000 and 320 000 cells/well; each dilution was distributed to 60 wells. The results show the minimum and maximum (mean±SD) number of positive wells for each dilution in responder (A and B) and non-responder (C) subjects.
Table 2.
Statistical comparison of wells positive for antibody to hepatitis B surface antigen (anti-HBs antibody) between responder and non-responder groups
| No. of EBV-infected PBMC/well | ||||
|---|---|---|---|---|
| Comparative groups | 320 000* | 160 000 | 80 000 | 40 000 |
| A & B | P < 0·05 | P < 0·02 | P < 0·05 | P < 0·05 |
| A & C | P < 0·005 | P < 0·005 | P < 0·005 | P < 0·02 |
| B & C | P < 0·0001 | P < 0·05 | P < 0·01 | P < 0·05 |
Epstein–Barr virus (EBV)-infected peripheral blood mononuclear cells (PBMC) were prepared at dilutions corresponding to 40 000, 80 000, 160 000 and 320000 cells/well; each dilution was distributed to 60 wells.
A, good responder; B, poor responder; C, non-responder.
Figure 2.
Limiting dilution assay of peripheral blood mononuclear cells (PBMC) for determination of the primary frequency of hepatitis B surface antigen (HBsAg)-specific B lymphocytes (minimum, maximum and mean) in healthy adult individuals responding to the hepatitis B vaccine.
Table 3.
Percentage of B lymphocytes in peripheral blood mononuclear cells (PBMC) of responder and non-responder vaccinees
| Subjects | Percentage of B cells |
|---|---|
| Good responders | 5–27* (14 ± 5) |
| (n = 34) | |
| Poor responders | 9–19 (14 ± 4) |
| (n = 5) | |
| Non-responders | 4–27 (16 ± 8) |
| (n = 6) |
The results show the minimum and maximum (mean±SD) percentage of CD19+ B cells in PBMC.
Table 4.
Frequency of hepatitis B surface antigen (HBsAg)-specific B lymphocytes in responder and non-responder subjects before and after hepatitis B vaccine
| After vaccination | |||
|---|---|---|---|
| Before vaccination | Good responders | Poor responders | Non-responders |
| < 1/49 115 | 1/387 to 1/44 460 | 1/7899 to 1/49 115 | < 1/49 115 |
| (1 : 6654 ± 1 : 7909)* | (1 : 28 505 ± 1 : 15 295) | ||
The results represent the minimum, maximum and (mean±SD) of B-cell frequencies obtained for responder and non-responder groups before and after vaccination.
Correlation between serum anti-HBs titre and HBsAg-specific peripheral blood B-cell frequency
A significant, positive correlation was observed between the mean serum titre of anti-HBs antibody and the frequency of circulating anti-HBs antibody-producing B cells in the responder group (r = 0·45, P < 0·005) (Fig. 3).
Figure 3.
Correlation between serum titre of antibody to hepatitis B surface antigen (anti-HBs antibody) and frequency of hepatitis B surface antigen (HBsAg)-specific B lymphocytes in healthy adult individuals responding to the hepatitis B vaccine.
Discussion
In the present study, EBV transformation and LDA were employed for the first time to estimate the frequency of HBsAg-specific B lymphocytes in hepatitis B vaccine responder and non-responder normal individuals. The methodology employed in this study has already been used by many investigators to enumerate specific B cells for a variety of antigens, including thyroglobulin, thyroid microsomal antigens, single-strand DNA (ssDNA), tetanus toxoid (TT), insulin,11 rabies virus9 and myelin basic protein (MBP).10 These studies reported diverse frequencies in the number of specific B cells, ranging from 1 × 10−2 for rabies virus9 to 0·7 × 10−5 for MBP.10 Two important points which might be overlooked by some investigators should be considered in such studies. First, the efficiency of transformation by EBV should be determined. EBV can infect all human B-cell precursors through its receptor CD21, but only a small fraction of the infected cells is transformed and immortalized. The proportion of transformed B cells varies from study to study and should therefore be determined in each individual study.13,14 In the present study, the efficiency of transformation was 3%, which is slightly higher than that obtained in our previous study13 and lower than that reported by others.14 The second point to be taken into account is estimation of the percentage of B cells in PBMC, if pure B cells are not used. This is important both for evaluation of the immune status of the subjects and for precise determination of the frequency of specific B cells. A low frequency of specific B cells may occur as a consequence of a low total B-cell count.
In our study, the total number of B cells was found to be similar in all responder groups (14%) and slightly higher in non-responders (16%) (Table 3), which argues against the above assumption.
Our results on the frequency of HBsAg-specific B cells in responders and non-responders, although not yet evaluated and confirmed, extends previous (mainly in vitro) studies indicating a lack or insufficient generation of specific B cells in HBsAg non-responder individuals.5–8 This could be either the result of a defect in the generation of a primary B-cell repertoire or insufficient T-cell help subsequent to HBsAg challenge in the secondary lymphoid tissues. The latter structural or functional defect could limit or inhibit the B-cell expansion phase, which usually occurs subsequent to antigen encounter prior to the differentiation pathway, leading to a diminished number of circulating specific B cells. The fact that in this study the HBsAg-specific B-cell frequency was the same before and after vaccination in non-responders and before vaccination in all responder groups (< 1/49 115) could point to the above assumption. The close correlation observed between the serum levels of anti-HBs antibody and circulating HBsAg-specific B cells in responder groups (Fig. 3), may also be regarded as evidence in support of this argument. Lack of or inadequate T-cell function may contribute to unresponsiveness to HBsAg at different cellular and molecular levels. Unresponsiveness to HBsAg vaccination has been repeatedly shown to be controlled by genes within the major histocompatibility complex (MHC).16–19 In humans, current data suggest considerable ethnic differences in the variety of human leucocyte antigen (HLA) genes associated with a lack of response to HBsAg.20 However, in most studies,18,19 as well as in our non-responder subjects,20 an increased frequency of HLA-DR7 and -DQ2 has been observed in non-responder individuals. Contribution of HLA antigens could influence the anti-HBs antibody response through selection of HBsAg-specific T-suppressor cells,17 inappropriate antigen presentation21,22 or induction of an unbalanced T helper 1/ T helper 2 (Th1/Th2) response.23,24 Induction of specific cytotoxic T lymphocytes (CTL), which could recognize and destroy HBsAg-specific B lymphocytes, has also been suggested.25
Primary or secondary defects in a specific Th lymphocyte repertoire may also be responsible. LDA analysis in responders and non-responders revealed marked discrepancies in HBsAg-specific T-cell precursor frequencies between responders and non-responders to the hepatitis B vaccine.23 None of the non-responders had any HBsAg-reactive T cells in the concentration ranges used (up to 5 × 104 T cells/well). The defect in specific T-cell repertoire could also be investigated by analysis of the T-cell receptor-Vβ (TCR-Vβ) genes expressed in responder and non-responder individuals. A recent report demonstrated increased frequencies of certain TCR-Vβ genes in HBsAg responder and non-responder vaccinees.26 The profile of the Th1/Th2 response and TCR-Vβ gene usage are currently being investigated in the responder and non-responder individuals in our laboratory to obtain further insight into the mechanisms involved in HBsAg non-responsiveness.
Acknowledgments
We wish to express our gratitude to Dr H. Mahmoodzadeh Niknam (from the Department of Immunology, Pasteur Institute of Iran) for providing the FACS facilities. We also acknowledge Dr A. Amirkhani (Department of Epidemiology), Dr Z. Samadi Bahrami, Dr A. Amanzadeh (National Cell Bank of Iran), and Dr A. Mirjalili (Department of Immunology) for their valuable co-operation.
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