| Background and aim:Occult HBV infection (OBI) defined as hepatitis B surface antigen (HBsAg) undetectable or negative in serum or plasma, but HBV DNA detectable in serum and/or liver tissue by sensitive in-house PCR is a peculiar status in HBV infection. However, the mechanism of OBI is unclear so far, maybe attribute to host immune response, viral mutation, host genetics and so on. Meanwhile, it is existed a phenomenon that HBsAg seroclearance or loss emerged in the patients recovered from chronic hepatitis B through antiviral therapy (interferon a or nucleot(s)ide analogue), which it is a small probability event in the treatment of chronic hepatitis B. Currently, it is still lack of investigation on the immune response in OBI without co-infection with other hepatitis virus. Nonetheless, the mechanism responsible for HBsAg seroclearance has never been explored in patients received antiviral therapy. Also, the characteristics of cellular immune response in patients with HBsAg seroclearance have not been demonstrated. In additional, it is very interesting that patients with either OBI or HBsAg seroclearance share the similar peculiarity, HBsAg undetectable or negative. Furthermore, whether the immune status in patients with HBsAg seroclearance through antiviral therapy was similar to occult HBV infection is also unknown. However, what type of immune responses would play a predominant role in eliminating HBsAg and virus? It has puzzled us for a long time. To demonstrate the possible mechanism responsible for occult HBV infection and HBsAg seroclearance, we have investigated the features of HBV-specific T-cell response in patients with either occult HBV infection or HBsAg seroclearance.Methods:(1)Nine hundred and eighty two blood donors were screened by a more sensitive chemiluminescent microparticle immunoassay for quantitative determination of HBsAg, and HBV DNA from the samples considered as reactive for HBsAg detection were extracted by QIAGEN MinElute Virus Spin kit, subsequently, the S, C, X and P gene were amplified by highly sensitive nested PCR using primers specific for different genomic regions and complementary to highly conserved nucleotide sequences. When no less than two regions were amplified positive, and ensured no contamination in the PCR system, it was diagnosed as occult HBV infection. Meanwhile, we analysed whether mutants were present in "a" determinant sequence, and HBV genotyping was also determined.(2) We have successfully followed up 6 blood donors with occult HBV infection, and found 3 patients from the out-patient and hospitalized with occult HBV infection. In parallel,14 patients resolved from chrionic hepatitis B with HBsAg seroclearance through effective antiviral therapy were enrolled in analyzing the characteristics of cellular immune responses. Furthermore, the HBV genotyping and virological analysis were performed in the patients with HBsAg seroclearacne.(3) The peripheral blood mononuclear cells (PBMCs) were isolated from 9 patients with occult HBV infection and 14 patients with HBsAg seroclearance, and stimulated by 18-mer overlapping peptide pool covered S and C-ORF, and recombinant HBV proteins (rHBsAg and rHBcAg). And then, we determined the production of interferon-γfrom specific T-cell by enzyme-linked immunosorbent spot (ELISPOT) assay. At the same time, the frequencies of interferon-y and interleukin-2 producing by HBV-specific T-cell response was analysed by intracellular cytokine staining, which stimulated by the overlapping peptide pools for 6 hours.(4) In additional,7 immunotolerant HBV carriers and 9 inactive HBsAg carriers were enrolled in this investigation as control, and the HBV-specific T-cell response was determined as mentioned before.Results:(1)We found HBsAg quantitive values in the plasmas from 64 blood donors were up the cut-off value (0.05IU/mL), which the positive rate was 6.5%(64/982). In these reactive samples,40 were male and 24 were female, but the mean age was no difference between male and female (27±6 VS.25±8,t=1.247,P=0.217). The mean of HBsAg quantitive level was different between HBsAg reactive and non-reactive (0.28±0.27 IU/mL vs.0.02±0.01 IU/mL; Z=-14.332,P<0.001).However, there was no difference in the ALT level between HBsAg reactive and non-reactive(15.3±5.8 VS.14.8±6.3,t=0.598,P=0.550).(2) HBV genomic fragments were amplified by sensitive nested PCR from 64 samples reactive to HBsAg quantitation. We successfully amplified S region in 19 samples (29.7%), C gene in 41 samples (64.1%),X gene in 42 samples (65.6%). Furthermore, some samples were just single region amplified, for example, one sample was positive for S region(1.6%),7 samples were positive for C region(10.9%) and 4 samples were positive for X region (6.2%).However, there were 39 samples amplified two or three fragments positive (60.9%). In detail,12 samples were identified for positive in triple fragments(18.8%),21 samples were positive for both C and X regions (32.8%), one sample were positive for both S and C regions(1.6%), 5 samples were positive for both S and X regions (7.8%). In addition,13 samples amplified were negative for any regions (20.3%).According to the diagnostic criterion of occult HBV infection,39 blood donors were diagnosed as occult HBV infection (4.0%,39/982).(3) In terms of sequence alignment, we did not find any important mutant in "a" determinant, especially the reported hotspot mutations in the fragment amplified from S gene in donors with occult HBV infection. Furthermore, HBV genotyping was analyzed in 14 donors with occult HBV infection through alignment, and 6 samples were considered as genotype B, and 8 samples were identified as genotype C. However, a 583bp fragment from the HBV open reading frame was amplified and sequenced from 6 of the 14 subjects with HBsAg seroclearance,5 of the 6 sequences were genotype C and one was genotype B. We found that two lamivudine-treated patients infected with a genotype C HBV developed L80I+M204I+L180M triploid mutants in the reverse transcriptase (RT) region.(4) HBV-specific T-cell response could be detected in patients with either HBsAg seroclearance or occult HBV infection through Elispot assay for interferon-gamma, which the frequencies of SFC in positive tests were ranged from 22 to 846 and 22 to 504 per million PBMCs respectively. However, the frequencies of positive tests were no difference between HBsAg seroclearacne and occult HBV infection (41.1% VS.33.3%,χ2=0.637, P=0.425), nor did the mean frequencies of SFC in positive tests between these two groups(113±36 vs.98±31,P=0.976). Meanwhile, the mean frequencies of SFC in positive tests from patients with either HBsAg seroclearance or occult HBV infection were higher than those in immunotolerant HBV carriers (P=0.031,P=0.040; respectively). In addition, HBV-specific T-cell response to rHBcAg was more dramatic than that against C peptide pool in patient with HBsAg seroclearance (P=0.012), in contrast, the HBV-specific T-cell response against rHBcAg or C peptide pool was no difference in patients with occult HBV infection (P=0.302). HBV-specific T-cell response against rHBcAg in patients with HBsAg seroclearance was stronger than those in occult HBV infection and immunotolerant HBV carriers (P=0.010, P=0.004), but similar to that in inactive HBsAg carriers (P=0.549). The immune responses in patients with HBsAg seroclearance were mainly to the C peptide pool and the rHBcAg (9 of 14 and 11 of 14 positive tests respectively). There were only three positive responses to S open reading frame antigens, and these were all close to the baseline response. Consistent with this, patients with HBsAg seroclearance displayed a more robust T-cell response against rHBcAg than against rHBsAg (P<0.001),and against the C peptide pool relative to the S peptide pool (P=0.001).The immune responses in occult HBV infection with seropositive for anti-HBc antibody were mainly to the C peptide pool (3/4), but they were hyporesponsive to S peptide, rHBcAg and rHBsAg. On the contrary, the immune responses in patients with seronegative occult HBV infection were mainly to the S peptide pool, C peptide pool and rHBcAg (3/5,respectively), but the magnitude of T-cell response in seronegative occult HBV infection was divergent. However, two patients with seropositive occult HBV infection (2/5) were positive response to rHBsAg, and they were both close to the baseline response.(5) Generally, Interferon-gamma producing HBV-specific CD4+ and CD8+T cells could be determined in peripheral blood mononuclear cells from patient with either HBsAg seroclearance or occult HBV infection. However, the frequency of IFN-y+CD8+T cells in patients with HBsAg seroclearance was similar to that in occult HBV infection (P=0.103), but the frequency of IFN-y+CD4+T in patients with HBsAg seroclearance was higher than that in occult HBV infection (P=0.047). The magnitude of HBV-specific CD8+T cell responses was significantly different among the groups we enrolled through integral comparison (χ2=9.417,P=0.024), and they displayed different responsiveness to S and C peptide pool. In detail, we found that the PBMCs from patients with HBsAg seroclearance was hyperresponsive to C peptide pool (78.6%,11/14; the frequency of IFN-y+CD8+T cells ranging from 0.06% to 0.24%, Median:0.10%), but lower response to S peptide pool (3/14, the frequency of IFN-y+CD8+T cells:0.08%,0.13% and 0.24%) and rHBsAg(1/14). Furthermore, there was no difference in the responsiveness to S (2/7, the frequency of IFN-γ+CD8+T cells:0.27% and 0.38%) and C peptide pool (3/7, the frequency of IFN-γ+CD8+T cells:0.08%-0.22%, Median:0.11%)in the occult HBV infection. In chronic HBV infection control groups, CD8+T cells from immunotolerant HBV carriers was hyporesponsive to S (3/7, the frequency of IFN-y+CD8+T cells: 0.06%～0.08%, Median:0.06%) and C peptide pool (2/7, the frequency of IFN-γ+CD8+T cells:0.06% and 0.07%), but in inactive HBsAg carriers, CD8+T cell responses to S and C peptide pool were similar (4/9, the frequency of IFN-γ+CD8+T cells:0.08%-0.32%, median:0.135%; 6/9, the frequency of IFN-γ+CD8+T cells: 0.06%-0.23%, median:0.085%, respectively).The magnitude of HBV-specific CD4+T cell responses was significantly different among the groups we enrolled through integral comparison (χ2=8.628, P=0.035), and they displayed different responsiveness to S and C peptide pool. In generally, the HBV-specific CD4+T cells responses to S peptide pool were no difference among all populations we investigated through integral comparison (χ2=4.241,P=0.237), in contrast, the HBV-specific CD4+T cells responses to C peptide pool were significantly different among four groups through integral comparison (χ2=2.881,P=0.005). Although there was no difference in the magnitude of CD4+T cell response in positive responses between S and C peptide pools in patients with HBsAg seroclearance (Z=-0.987, P=0.324), they differed in the magnitude of CD4+T cell response in overall response (Z=-2.151,P=0.031).And the positive rates were different between S and C peptide pool (4/14 vs.11/14) in CD4+T cell response from patients with HBsAg seroclearance. Patients with occult HBV infection displayed a lower CD4+T cell response to S and C peptide pool (2/7, the frequency of IFN-y+CD4+T cells:0.06% and 0.07%; 3/7, the frequency of IFN-γ+CD4+T cells:0.06%,0.07% and 0.10%), and there were no difference in the magnitudes of CD4+T cell response in either overall response or positive response between S and C peptide pool (Z=-0.355,P=0.723;Z=-0.609, P=0.543). CD4+T cells from immunotolerant HBV carriers hardly responded to C peptide pool (positive test: 0/7), but responded to S peptide pool in a low frequency (3/7, the frequency of IFN-γ+CD4+T cells:0.06%,0.06% and 0.07%). The magnitude of CD4+T cell response against C peptide pool in overall responses from patients with HBsAg seroclearance was more vigorous than those in occult HBV infection and immunotolerant HBV carriers (Z=-2.254,P=0.024;Z=-3.257,P=0.001),but was similar with that in inactive HBsAg carriers (Z=-0.823,P=0.411).However, the magnitude of CD4+T cell response against C peptide pool in overall responses from occult HBV infection was similar with those in immunotolerant HBV carriers and inactive HBsAg carriers (Z=-0.067, P=0.947; Z=-1.763,P=0.078). CD4+T cells from occult HBV infection with seropositive to anti-HBc antibody rarely responded to S and C peptide pool, only one test was positive reactive to S peptide pool in CD8+T cell response. On the other hand, occult HBV infection with seronegative to anti-HBc antibody displayed CD4+T cell response to S and C peptide pool (2/4 and 3/4 respectively), and sod did the CD8+T cell response(1/4 and 3/4, respectively). However, the magnitude of T cell response against C peptide pool was no difference between CD4+T cell and CD8+T cell in seronegative occult HBV infection (Z=-1.528,P=0.127).(6) We found that the T cell response against S or C peptide pool was heterogeneity in all subjects when the IL2 producing T cells were determined by intracellular cytokine staining. Patients with HBsAg seroclerance displayed a low responsiveness to S and C peptide pool in CD8+T cell response (4/14 and 2/14) and CD4+T cell response (both 1/14). The similar responsiveness was found in CD4+T cell (S:2/9, C:1/9) and CD8+T cell (S:2/9, C:3/9) in inactive HBsAg carriers. However, patients with occult HBV infection showed a low reactiveness to S peptide pool in CD4+T(1/7) and CD8+T cell (2/7) responses, but rarely responded to C peptide pool in CD4+T and CD8+T cell responses (both 0/7). Surprisingly, immunotolerant HBV carriers displayed a relatively high responsiveness to S peptide pool in CD4+T (3/7) and CD8+T cell (4/7) responses, but a low reactiveness to C peptide pool in both CD4+T and CD8+T cell responses (both 1/7). Furthermore, we found there was significantly difference in the frequencies of IL2+CD8+T cell among all populations we investigated (χ2=8.269,P=0.041),but no difference was found in the frequencies of IL2+CD8+T cell (χ2=2.188,P=0.534), when integral comparison was performed by Kruskal-Wallis H test.Conclusions:There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects. Occult HBV infection displayed a different pattern of immune responses in seropositve and seronegative. Although HBsAg negative or undetectable was shared by patients with either occult HBV infection or HBsAg seroclearance, we thought that the responsible mechanisms seemed to be divergent. According to the features of cellular immune responses and the natural history of chronic HBV infection, we considered that occult HBV infection would mainly come from the patients with resolved acute hepatitis B, yet patients with HBsAg seroclearance may be under inactive immune phase or recovery phase. Nonetheless, they might be reversed to HBV relapse caused by immunecompromised or viral breakthrough. Therefore, a long-term follow-up would be necessary for patient with HBsAg seroclearance through antiviral therapy. Meanwhile, combined with sensitive HBsAg assay and screening anti-HBc antibody or nuclear acid test would reduce the risk of occult HBV infection in transfusion or organ transplantation.
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