In this issue of the journal, Dr. Luo and co-workers have identified intra-graft hepatitis B virus (HBV) specific lymphocytes in HBV immune liver donors, (most of whom were live donors) and correlated their findings with the liver transplant recipients' humoral immune responses to HBsAg.1 HBV specific T and B cells were identified in 59% and 28% of liver grafts respectively in 32 donors. After liver transplantation, almost half of the transplanted patients with chronic HBV developed anti-HBs seroconversion which correlated with the number of donor graft derived HBsAg specific T cells. The investigators suggest that these donor derived HBV specific lymphocyte may be the vehicle through which adoptive transfer of immunity is transferred from HBV immune donors to liver graft recipients with persistent HBV infection. This important study provides for the first time evidence on HBV specificity of intrahepatic lymphocytes isolated from human liver grafts and transplanted to patients with chronic HBV infection. The study sheds further light on the presumed mechanism involved in adoptive transfer of immunity in the setting of parenchymatous organ transplantation. Furthermore, it may take us one step forward in the quest for developing new means for the so called “vaccinotherapy” for controlling persistent HBV infection.
Analysis of the reported data requires a short look back on the progress already achieved in this exciting area of clinical research.
Patients with acute HBV infection who recover spontaneously are able to clear HBV from the circulation by mounting an effective innate, cellular and humoral immune response leading to long-term, usually life-long immunity.2, 3 Some patients may clear the virus from the circulation, but remain with occult HBV infection with covalently closed circular (ccc) HBV-DNA “hiding” in hepatocytes. Yet, these recovered patients rarely reactivate HBV infection in the presence of an intact immune system and “immune memory” which serve as a gate keeper for prevention or suppression of viral replication and its consequences. In contrast, patients with persistent HBV infection are often tolerant to HBV and unable to mount an effective cellular and humoral immune response toward nucleocapsid and envelope proteins of the virus. This is also true for HBV patients undergoing liver transplantation who in addition to this immunologic defect are also immune suppressed by pharmacologic agents for prevention of graft rejection.
Current strategies for protection of HBV liver transplant recipients at risk include lifelong monotherapy with hepatitis B immune globulin (HBIG) or a nucleoside analogue or combination therapy using the two agents simultaneously and indefinitely or at least for a defined period, followed by life-long monotherapy.4 These strategies, which also include pre-transplant suppression of HBV viral load, have led to an impressive success rate in prevention of HBV graft re-infection in about 90% of patients treated with combination therapy. However, although the risk of graft re-infection diminishes over time in treated transplant recipients, complete eradication of HBV and elimination of infected hepatocytes containing cccHBV-DNA still remains an elusive goal. Furthermore, the different means for prevention of HBV graft re-infection have several limitations including high cost, the constant need for expert supervision, the inconvenience for patients leading to reduced compliance and the risk (albeit rare) of generating HBIG induced HBsAg mutants. Thus there are good arguments for exploring new means for immunotherapy for HBV transplant patients which should contain HBV activity through generating an effective immune response, preventing HBV re-infection of the graft or reactivation of occult infection.
Indeed, proof for the potential application of immunotherapy in such patients can be derived from a number of observations which suggest that immune memory against HBV nucleocapsid and envelope protein(s) can be adoptively transferred from organ donors to recipients. Initially, the Jerusalem group was able to document clearance of HBV from the circulation in an HBsAg+/anti-HBe+/HBV-DNA+ bone marrow transplant patient with chronic lymphocytic leukemia who received an HLA matched bone marrow from his anti-HBc+/anti-HBs+ brother.5 Adoptive transfer of immunity to HBsAg was than shown to occur in HBV naïve bone marrow or peripheral lymphocytes and stem-cell recipients from immunized anti-HBs+ donors.6, 7 Immune memory to HBsAg could be boosted with an HBV vaccine in such patients.8 Dr Lau and co-workers have confirmed and extended these observations in HBsAg+ transplant recipients in Hong-Kong in whom HBV clearance in patients receiving an HBV immune bone-marrow (anti-HBc+/anti-HBs+) was associated with ALT elevation followed by anti-HBs seroconversion.9 Furthermore, donor derived core antigen reactive T-cells were linked by these investigators to clearance of HBV in bone marrow transplant HBsAg+ patients.10
A number of clinical studies have than provided further evidence that immunization of bone marrow, lymphocyte and stem-cells donors against HBV may induce HBV immunity in transplant recipients who were capable of mounting an anamnestic anti-HBs response to a booster vaccination.11–13 Studies in animal models developed in mice14, in rats15 and in woodchucks16 confirmed that indeed immunity to HBV can be adoptively transferred even under conditions of immune suppression employed in organ transplantation. This was also reported in experimental heart and kidney transplantation.17 Finally, in the woodchuck model, liver transplantation from woodchuck hepatitis virus immune donors to woodchucks with persistent woodchuck hepatitis virus infection enabled control of the viral infection in transplanted animals.16 Thus, results obtained through clinical observations in humans and experiments in woodchucks suggest that clearance of persistent HBV infection is attainable through adaptive transfer of immunity to HBV from healthy donors to immunosuppressed recipients. Sette et al. have already shown that T cell tolerance to HBV proteins can be broken with appropriate immunization in transgenic mice.18 The overall results from the studies cited here provide a rational for developing new means of immunotherapy for HBV including “vaccinotherapy” which so far has met with limited success.19 Until now, recurrent attempts to immunize HBV liver transplant patients against HBV have also met with rather marginal success except for a few exceptions.20–22 However, the introduction of a novel adjuvant in combination with recombinant yeast-derived HBsAg particles has been recently reported to induce a potent anti-HBs response in immune suppressd HBV liver transplant patients.23
Following the reports cited here, it became clear that transplanted organs from HBV immune donors may contain HBV lymphocytes which react against nucleocapsid and/or envelope proteins. However it was unclear whether this phenomenon reflects passive transfer of immune lymphocytes from HBV immune donors to graft recipients. Alternatively it could be either the result of engraftment of such donor lymphocytes in the transplant recipients with active generation of HBV specific cellular and humoral immune responses or the result of chimerism between donor and recipients lymphocytes.
A recent report from the Hong-Kong group generated much enthusiasm and hope that use of anti-HBc+/anti-HBs+ grafts will generate prolonged anti-HBs production in HBV liver transplant recipients, thus eliminating the need for HBIG coverage.24 In their report, Dr Luo and co-workers observed spontaneous anti-HBs seroconversion in 42% of 50 patients receiving lamivudine monoprophylaxis after liver transplantation from HBV immune donors (about two thirds of whom were living, related donors). Seroconversion to anti-HBs occurred in liver graft recipients at a median interval of 8 days post transplantation (range 1-43 days), similar to the observation in bone marrow transplant donor and recipient pairs.6 Anti-HBs titers increased progressively to a maximum level at 3 months post transplantation. The pace of hepatitis B surface antigen (HBsAg) clearance from the circulation of transplanted patients correlated with their anti-HBs titer. The investigators postulated that the combined effect of lamivudine which down regulates HBV replication and the presence of circulating anti-HBs have lead to clearance or at least suppression of HBV in these patients. These preliminary short-term results provided support to the hypothesis that donor derived HBV immune lymphocytes may indeed engraft in the recipient which were able to mount a protective anti-HBs response. The relatively rapid drop of anti-HBs titers in transplanted patients did not discourage these investigators since this is a common phenomenon after immunization, especially in immune-suppressed patients. It was hoped that HBsAg specific B cell memory can be boosted and revived by an HBV vaccine as observed in healthy vaccinees.25 However, this hope eventually turned out to be premature. Active immunization of these transplanted patients with two courses of a double dose HBV vaccine did not lead to an anamnestic rise in anti-HBs titers in the majority of patients.26 Thus, adoptive transfer of immunity to HBV in liver transplant recipients may be a temporary phenomenon with no permanent engraftment of immune lymphocytes in transplant recipients. This stands in contrast to observations in BMT recipients in whom immune memory to HBV could be boosted post transplantation.8 How can this difference in success rate in post transplantation revival of immune memory against HBV between bone marrow transplant and liver graft recipients be explained?
The ultimate goal of adoptive transfer of immunity in HBV liver transplant patients is the long-term maintenance of plasma anti-HBs levels post transplantation, preferably above 100 IU/L. The present study of Dr. Luo and co-workers provides new insight into the putative mechanism of adoptive transfer of immunity to HBV in the liver transplant setting.1 It also gives indirectly a possible clue as to the reason for the partial failure of post transplantation immunization against HBV in patients in whom short-term adoptive transfer of immunity has been successful.
In their new study1 the Hong Kong investigators provide proof for the presence of HBsAg and hepatitis B core antigen (HBcAg) specificity of T-cells recovered from liver grafts of HBV immune donors. They also present evidence on the correlation between HBsAg specific B cells in donors and anti-HBs production in liver transplant recipients. Interestingly, although HBV specific T and B cells were detectable in 59% and 28% respectively of donor grafts, the number of detectable HBsAg-specific, HBcAg specific T cells as well as anti-HBs secreting B cells in the graft (before transplantation) was relatively low. Too small a number of engrafted HBV immune lymphocytes may be a reason or one of the causes why anti-HBs levels decline rapidly in these liver transplant recipient who do not respond adequately to booster HBV vaccination (despite successful adoptive transfer of immunity). Apparently, in bone marrow transplant patients who receive an HBV immune bone marrow, enough HBV immune lymphocytes are being transferred to enable engraftment of a larger population of donor cells which may respond to a booster vaccination with an anamnestic rise in anti-HBs titers.
In their study, Luo et al. have shown that adoptive transfer of immunity to HBV patients undergoing liver transplantation was most successful, when the donor serologic profile was anti-HBc+/anti-HBs+. Yet, an almost similar success rate could be obtained using liver grafts from anti-HBc−/anti-HBs+ donors who were previously immunized against HBV. In theory, utilization of an anti-HBc+/anti-HBs+ graft donor should have an advantage in comparison to an anti-HBc−/anti-HBs+; donor since clearance of HBV infection requires both core and envelope protein specific T cells as shown in the bone marrow transplant setting.5, 9 However, the situation in liver transplant recipients is unique, since adequate anti-HBs levels alone can contain HBV and prevent HBV reinfection of the graft in patients at risk.4 Therefore, utilization of liver graft donors who have been immunized against HBV may still be effective provided these patients' adoptively transferred immune memory against HBV can be boosted. Evidently, the currently available licensed yeast derived HBV vaccines formulated with alum hydroxide, are unable to provide the required boost,22 possibly due to their relative low immunogenicity in immunesuppressed patients as well as to the small number of engrafted HBV immune lymphocytes. Indeed, Dahmen et al. were able to show in an experimental model that the efficiency of humoral immune transfer depends on both the graft and the recipient's number of lymphocytes.27 More potent HBV vaccines containing new adjuvants or PreS/S (HBsAg envelope proteins including small-S, middle-Pre S2, and large-Pre S1 antigens) epitopes have recently been reported to induce impressive anti-HBs responses in vaccine non-responders and immune suppressed patients.23, 28, 29 Such vaccines may expand the number of HBsAg specific resident lymphocyte clones in patients in whom successful adoptive transfer of immunity against HBV envelope epitopes has been achieved. At this stage, there is no evidence that adaptive transfer of immunity against HBV in the setting of liver transplantation provides long-term immunity against HBV. Therefore, currently, it appears as if this maneuver results in a transient immunologic phenomenon.
However, the cumulative data obtained from observations in bone marrow transplantation as well in liver transplant recipients suggest that immunotherapy in patients with chronic HBV infection, including liver transplant patients may after all have some merits. This assessment is based on a number of observations in animals and humans and despite relatively low success rates of the so called “vaccinotherapy” using conventional HBV vaccines.19 The key factor in obtaining this difficult goal depends on the efficient induction of specific T-cell responses in graft recipients against nucleocapsid and envelope proteins of the hepatitis B virus. This has already been achieved in bone marrow transplant recipient transplanted from anti-HBc+/anti-HB+ donors.5, 9, 11, 12 Furthermore, previous results also suggest that adoptively transferred immunity to HBsAg alone, even in the absence of HBcAg specificity, may be sufficient to contain HBV infection in bone marrow recipients at risk.
The study by Luo and co-workers still requires some extension since it only provides information on the overall HBV reactivity of HBV immune lymphocytes isolated from graft donors without defining epitope specificities for core and envelope proteins. Furthermore, for obvious technical reasons, no information is provided regarding the same T and B cell specificities isolated from grafts after transplantation. Also, it would be interesting to know if the adoptively transferred immunity to HBV is generated by engrafted HBV immune lymphocytes or/and through chimerism between donor and host lymphocytes.
In summary, the study of Dr. Luo and co-workers adds new information on the role of intra-graft lymphocytes with anti-HBV specificity in adoptive transfer of immunity against HBV in liver transplant patients. It also takes us one step further on the bumpy road for development of new means for immunotherapy in patients with persistent HBV infection.