aLaboratoire de Virologie, Hôpital Lapeyronie, Montpellier, France
bINSERM U847, Montpellier, France
cDepartement des Maladies Infectieuses et Tropicales, Hôpital Gui de Chauliac, Centre Hospitalier Universitaire de Montpellier, Montpellier, France.
Received 13 April, 2007
Accepted 30 April, 2007
In HIV-1-infected patients, although serological evidence of previous exposure to hepatitis B virus (HBV) is frequent  and vaccination against HBV widely recommended , a fast decline of hepatitis B surface antigen (HBsAg) specific antibodies (anti-HBs antibodies) has been reported after HBsAg vaccination or HBV recovery [3,4]. Moreover, the serological pattern of anti-hepatitis B core antibody positive (anti-HBc+, anti-HBs−, and HBsAg−) also called isolated anti-HBc seropositivity, is common among HIV-1-infected patients who have recovered from HBV [1,5]. Circulating HBV-specific T cells have recently been identified in HIV-1-infected patients who have resolved HBV infection , whereas the presence of HBsAg-specific memory B cells has never been reported. Serum antihepatitis B surface antibodies could be undetectable in some healthy individuals vaccinated against HBV, whereas memory B lymphocytes against HBsAg are present [7,8]. Therefore, in this work we have enumerated HBsAg-specific memory B cells in HIV-1-infected patients with either natural or vaccinal immunity to HBV and in HBsAg-vaccinated controls. HIV-1-specific memory B cells were concomitantly enumerated to explore a possible association with HBsAg-specific memory B cells.
Twenty HIV-1-infected patients and 20 healthy controls were enrolled after providing written informed consent. All patients received antiretroviral therapy (ART). The first group of HIV-1-infected patients consisted of 10 HBsAg-vaccinated individuals without serological evidence of HBV exposure (anti-HBc−). The second group consisted of 10 HIV-1-infected patients who had recovered from HBV infection and had no recent history consistent with symptomatic acute hepatitis B nor biological markers of HBV viral replication (anti-HBc+, HBsAg−, HBV-DNA−), and the date of HBV acute infection was known for only one case (patient R4). The clinical and biological characteristics of HIV-1-infected patients are given in Table 1. HBsAg-vaccinated controls had a median duration after the last HBsAg vaccination of 10 years [interquatile range (IQR) 4–18] and a median serum anti-HBs antibody level of 145 mIU/ml (IQR 87–1020).
Approximately 1–2 million circulating B cells were purified by depleting unwanted cells from ethylenediamine tetraacetic acid-treated blood samples using a RosetteSep B cell enrichment cocktail (Stemcell Technologies, Meylan, France). B cells were stimulated with CD40L-expressing CDw32L mouse fibroblasts plus IL-2 and IL-10 (Tebu, Le Perray en Yvelines, France) as previously described . The efficiency of memory B cell polyclonal activation was controlled by the enumeration of immunoglobulin secreting cells (Ig-SC) using a κ + λ-L chain ELISpot assay. Cells secreting antibodies directed to HBsAg (HBs-SC) or HIV-1 antigens (HIV-1-SC) were detected by a two-colour ELISpot assay adapted from hepatitis B surface and HIV-1 single-colour ELISpot assays using alkaline phosphatase-conjugated HBsAg (Adw + Ayw) and horseradish peroxidase-conjugated HIV-p24 and -gp41 antigens, kindly provided by Dr Delagneau (Bio-Rad Marnes-la-Coquette, France), as previously described [8,9].
The median values were compared using the Mann–Whitney U test. Correlations between variables were tested using linear regression and Spearman rank tests. P < 0.05 was regarded as significant.
We tracked HBsAg-specific memory B cells in HIV-1-infected patients with either natural or vaccinal immunity to HBV. HBs-SC were found in nine out of 10 HBsAg-vaccinated and in nine out of 10 HBV naturally immunized patients (Table 1). HBs-SC represented 0.004–0.52% of the total Ig-SC (median 0.09; IQR 0.008–0.27). HBs-SC of the IgG isotype were found in 14 out of 18 HIV-1-infected patients, IgA in 11 out of 18 and IgM in 12 out of 18. A median number of 18.5 HBs-SC/106 B cells were found in vaccinated patients and 50.5 HBs-SC/106 B cells (P = 0.16) were enumerated in HBV recovered patients. In two patients having undetectable antihepatitis B surface antibodies in serum, we did not detect HBs-SC despite good polyclonal activation as controlled by Ig-SC (data not shown), suggesting a lack or a shortage of HBsAg memory B cells. HBs-SC were enumerated for all the vaccinated controls, median 93 HBs-SC/106 B cells (IQR 15–135), corresponding to 0.002–0.27% of the Ig-SC (median 0.028; IQR 0.003–0.29). The frequency of these cells was greater for vaccinated controls than for vaccinated HIV-1-infected patients (P = 0.018). HBs-SC numbers were similar in vaccinated controls and HBV immunized patients (P = 0.39).
HIV-1-SC were also found in 15 out of 20 patients (Table 1), median 30 HIV-1-SC/106 B cells, and corresponded to 0.003–0.68% of the Ig-SC (median 0.28; IQR 0.002–0.085). The numbers of HBs-SC and HIV-1-SC were compared and no correlation was found (r = −0.11; P = 0.64).
Our results demonstrated that HBsAg-specific memory B cells could be detected several years after HBsAg vaccination or HBV recovery in HIV-1-infected patients including some individuals with undetectable anti-HBs antibodies in serum. These results suggest that the HBsAg-specific memory B cell population is better sustained than anti-HBs antibodies in serum. Nevertheless, the generation or longevity of HBsAg memory B cells after vaccinal immunization seems impaired in HBsAg-vaccinated patients and the lower number of hepatitis B surface-specific memory B cells detected compared with controls support this hypothesis. Our results are in agreement with others showing a lower number of memory B cells specific to measles, Streptococcus pneumoniae, and influenza virus in HIV-1-infected patients compared with controls [10,11].
HIV-1-infected patients who had resolved HBV infection exhibited a higher number of HBs-SC than vaccinated patients, although the difference did not achieve statistical significance, and a similar number of HBs-SC compared with vaccinated controls. In HIV-1-uninfected subjects, we and others have previously reported a comparable number of HBs-SC after vaccination or HBV recovery, suggesting that the two processes have equivalent efficiency to generate persistent HBsAg-specific memory B cells [8,12]. Accumulated data argue in favour of persistent interactions between specific immune cell effectors and residual HBV antigens in patients who have recovered from HBV [13,14]. In HIV-1-infected patients recovered from HBV the long-term maintenance of memory B cells specific for HBsAg may thus partly result from the de novo generation of these cells in response to residual HBV antigen stimulations.
The lack of correlation between the numbers of HIV-1 and HBsAg-memory B cells did not support the hypothesis of a relationship between the two memory B cells populations. Regarding HIV-1 infection, there is numerous evidence of B cell residual HIV-1 antigen exposure in good responders to ART [15–17]. Nevertheless, HIV-1-specific memory B cells could be difficult to detect or disappear in some patients on ART . These data argue for a short half-life or a poor generation of HIV-1-specific memory B cells.
Therefore, HBsAg memory B cells persisted in HIV-1-infected patients, even when the anti-HBs antibody level was below 10 mIU/ml. In these patients protection against clinically significant breakthrough hepatitis B may depend on the ability of specific memory B cells to proliferate rapidly and differentiate into plasma cells in response to HBsAg.
The authors would like to thank Marie-France Huguet and Karine Bolloré for expert technical assistance. They also thank Sharon Lynn Salhi for presubmission editorial assistance and Jacques Ducos for helpful discussion.
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