Front. Med. 2014, 8(1): 17–23 DOI 10.1007/s11684-014-0313-7

REVIEW

Vaccine therapies for chronic hepatitis B: can we go further? ✉)1, Xuanyi Wang1,2, Bin Wang1, Zhenhong Yuan1

Yumei Wen ( 1

Key Laboratory Medical Molecular Virology of Ministry of Education/ Ministry of Health, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2014

Abstract Chronic hepatitis B is a major health burden worldwide. In addition to the recent progress in antiviral treatment, therapeutic vaccination is a promising new strategy for the control of chronic hepatitis B. On the basis of the major specific and non-specific immune dysregulations and defects in chronic hepatitis B patients, this paper presents the peptide and protein-based, DNA-based, cell-based, and antigen-antibody-based therapeutic vaccines, which have undergone clinical trials. The advantages, disadvantages, and future perspectives for these therapeutic vaccines are discussed. Keywords

chronic hepatitis B; therapeutic; antigen-antibody complexes; DNA; vaccine

Introduction Despite the significant contribution of preventive hepatitis B vaccine in reducing the incidence of hepatitis B virus (HBV) carriers worldwide, approximately 300 million people are still infected with HBV. Some of these infections progress to chronic liver diseases, liver cirrhosis, and even hepatocellular carcinoma. Antiviral treatment for chronic hepatitis B (CHB) patients has undergone considerable improvement during the past decade. However, both antiviral therapy and immunotherapy are needed for patients with persistent infections such as CHB. Antiviral therapy can be regarded as “direct therapy” because its effects directly target the virus to inhibit viral replication and expression of viral proteins, thereby reducing inflammation or other damages in the host. However, the virus and virus-infected cells may not be eliminated. By contrast, immunotherapy can be regarded as “indirect therapy” because it triggers host immune responses to repair the immunologic defects and supplement necessary immune factors to inhibit or clear the persisting virus and virus-infected cells. Responses to antiviral therapy are relatively rapid, but for persistent viral infections, long-term treatment is necessary. In addition, emergence of drugresistant mutants and safety of long-term antiviral drug use should be considered. Another important issue is the high cost of antiviral drugs, which brings considerable financial burden to the patients or to the health-care system. Contrary to

Received August 21, 2013; accepted November 6, 2013 Correspondence: [email protected]

antiviral therapy, responses to immunotherapy vary among patients because the therapeutic efficacy depends on the status of innate and adaptive immunity in patients. In vaccine therapy, different subpopulations of patients may require different therapeutic immunization protocols. Given that a time lapse exists between immunization and immune response development, delayed efficacy in therapeutic vaccination is expected. Therefore, the risk of occurrence of dysregulation in host immune responses, such as autoimmune responses, should be considered. Antiviral and immunotherapies are two interrelated approaches that should be carefully implemented to achieve synergy. However, negative effects of the combined approaches should also be considered.

Major innate and adoptive immune disorders in CHB patients In general, innate responses are crucial for early viral containment and efficient induction of virus-specific adaptive responses, whereas adaptive immune responses are necessary for complete and persistent control of infections. Defects in natural killer (NK) cells and Kupffer cells (KCs) During the early phase of HBV infection, activating innate immunity, particularly NK and NKT cells which produce interferons (IFNs), are important factors. Defects in NK cell activation and reduced antiviral cytokine secretion would result in failure in viral containment and subsequent induction

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of effective adaptive immune responses [1]. KCs are longlived and abundant macrophages in liver with multiple immunologic functions, and they represent 15% to 20% of the total live cell population. Innately activated KCs upregulate adaptive responsive genes and promote primed T cell activation [2]. Defects in KCs and reduced Toll-like receptor 2 (TLR2) expression, are found in HBV infections, such as in HBeAg-positive HBV infection [3]. Additionally, in the presence of HBV, particle-activated macrophages produce less TNF-α in vitro when exposed to lipopolysaccharide (TLR4 ligand). Defects in dendritic cells (DCs) DCs are classified into two major subsets, namely, myeloid and conventional DCs. Conventional DCs are potent antigenpresenting cells, whereas plasmacytoid DCs (pDCs) recognize viruses through the sensing of viral nucleic acids, single stranded RNA, and unmethylated DNA motifs via TLR7 and TLR9 [4]. Large amounts of type I IFN and proinflammatory cytokines are subsequently produced, thereby inhibiting viral infection and modulating both innate and adaptive antiviral immunity [4]. In CHB infections, although the frequencies of circulating and intrahepatic pDCs between patients and noninfected controls are similar, the co-activation molecules CD40 and CD86 are overexpressed in CHB patients compared with noninfected controls. Significantly lower expression of OX40L on pDCs, which is closely correlated to viral load and hepatitis B surface antigen (HBsAg) concentration, is also found in CHB patients. Impaired OX40L expression by pDCs is responsible for the failure of pDCs to activate NK cell cytotoxicity. In addition, high levels of plasma IP-10 in chronic HBV patients are also incriminated for functional DC defects [5]. Roles and specific defects in CD4 and CD8 T cells Viral specific T cells are crucial in controlling virus infections, but they simultaneously cause inflammatory pathogenesis. T cell frequency and function is significantly superior in patients with resolved HBV infection than those with CHB infection. T cells also produce antiviral cytokines (IFN-γ and TNF-α) to clear HBV from infected hepatocytes without extensive direct killing of these cells. However, CD4+ and CD8+ T cells function differently in HBV infections. In chimpanzee model, CD4+ T cell depletion prior to HBV infection results in the development of persistent infection in the absence of a detectable CD8+ T cell response. However, no effect on viral clearance or liver disease is found when CD4+ T cells are depleted at the peak of infection, whereas virus-specific CD8+ T cells are detectable [6]. On the basis of the experimental and clinical results [7], HBV-specific CD4+ T cells do not primarily mediate direct antiviral effects, but they are important in the clearance of the virus by enhancing the effector responses of virus-specific CD8+ T cells. The

Therapeutic vaccine for chronic hepatitis B

assistance provided by CD4+ T cell is important in the induction and maintenance of virus-specific CD8+ T cell response, which can clear the virus by cytolytic and noncytolytic effector mechanisms. High viral load would inhibit HBV-specific CD8+ T cells, which are hardly detectable in patients with viral loads >107 copies/ml. Exhaustion of T cells, which may be caused by extrinsic and intrinsic mechanisms, is important in defects of specific T cell responses. As an intrinsic effect, the pro-apoptotic molecule Bim is specifically upregulated in HBV-specific CD8+ T cells obtained from chronically infected patients [8]. Another intrinsic factor is the increased expression of the inhibitory receptor programmed death-1 (PD-1) on HBVspecific CD8+ T cells in CHB patients, which may contribute to CD8+ T cell dysfunction and apoptosis [9]. High viral load or high viral proteins in hosts are important extrinsic factors which cause impaired T cell functions. Other factors generate a tolerogenic environment, which consists of immunosuppressive cytokines and regulatory T cells. KCs that express the immunosuppressive cytokines interleukin-10 (IL-10) and transforming growth factor β (TGF-β) are involved in the generation of a unique cytokine environment, which induces tolerance of liver-infiltrating lymphocytes in mice [10]. Furthermore, increased amount of regulatory T cells characterized by the expression of FoxP3 and CD25 is important in immune tolerance to CHB [11]. Disorders in specific antibodies Given the immune defects in T cell responses, antibodies against HBsAg are rarely detected in CHB patients, whereas antibodies against HBcAg are usually present, because production of anti-HBs is T cell dependent, whereas antiHBc is both T cell dependent and T cell non-dependent [12]. Immune complexes of HBsAg, HBeAg, and Pres-S [13] have been detected in HBV-associated glomerulonephritis. These complexes are considered to be related to the relative antigen antibody ratio, as well as the size, valence, and nature of the antigen, and the class and affinity of the antibodies. Low affinities of anti-HBs and anti-HBc have been reported in CHB patients [14], and coexistence of HBsAg and anti-HBs in some patients has also been described. The anti-HBs in such patients may be specifically directed to HBsAg subtypes, which are different from the coexisting HBsAg. Low affinity of anti-HBs, and lack of neutralizing effects of the coexisting anti-HBs may indicate defects in antibodies in these patients [15]. The immune defects/disorders reported in CHB are summarized in Fig. 1.

Types of immunotherapies for CHB patients To restore the immune defects/disorders in CHB patients, methods to revert the immune disorders have been developed, which may be categorized as follows:

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Fig. 1 Mechanisms of immune disorders in CHB. In this diagram, the immune disorders in the liver tissue of CHB patients are divided into innate and acquired immunity. Defects in NK cells, DCs, and KCs are shown as defects in innate immunity, whereas defects in APCs, CD8 T cells, and immune inhibitory cells (Treg), and inhibitory PD1 are shown as defects in acquired immunity. Details of these defects are listed in the text.

Immune stimulation (therapeutic vaccination) is performed by active immunization to stimulate effective immune responses in infected patients. The efficacy of this type of immunotherapy is mainly determined by the immune status of the patients. The innate and adaptive immune responses of the treated patients should at least be able to respond to manipulation with immune stimulants, otherwise this approach is ineffective. For active immunization, the timing and course of treatment are crucial and should be carefully investigated. The efficacy of active immunization is relatively long-term, and it needs boosting at appropriate intervals. Immune supplementation is performed by passive immunotherapy to provide patients with cells or antibodies to restore effective immune responses in patients. This approach includes infusion of engineered cells or cells prestimulated with proteins or pre-incubated with cytokines [16,17] and direct infusion of antibodies. IFN treatment can also be classified in this category, because IFN not only has antiviral effects but can also modulate host immune responses [18–20]. Effective innate and adaptive immune responses in patients are not required in this type of treatment. However, continuous multiple courses of treatments are necessary for passive immunotherapy, and the chances of complete

restoration of effective immune responses are low. Immune moderation is performed by blocking negative regulatory cells or factors, such as PD-1 [21], PD-1 ligands, or with the use of antibodies against IL-10 [22], or by providing positive immuno-cytokines, such as IL-2 and IL-12, to restore effective immune responses in patients. These approaches which target negative regulatory mechanisms or positive regulators may reshape the immune responses in patients and result in effective treatment. However, individual cases may respond differently, and potential ill effects caused by immune dysregulation should be avoided.

Types of therapeutic vaccines for CHB patients Herein, we focus on the immune stimulation approach, which aims to restore effective immune responses by active immunization. Four types of therapeutic vaccines are currently available based on different factors which have undergone clinical trials to stimulate effective immune responses in CHB patients. The advantages and disadvantages of these approaches are summarized in Table 1.

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Therapeutic vaccine for chronic hepatitis B

Table 1 Advantages and disadvantages of the approaches in activating immune responses in CHB patients Technologies

Advantages

Disadvantages

Protein- or peptide-based

Induces high titers of antibody response with specificity, easy to produce, and can be combined with other agents for coadministration

Large doses required, relative weak cell-mediated responses, and needs adjuvant and repeated administrations

Antigen-antibody complex-based

Induces specific immune response, enhances uptake by APC via Fc receptors, enhances DC-T cell interactions, breaks immune tolerance, and activates complement reactions

Need to optimize the ratio of antibody and antigen and antibody species should match with the recipient

Antibody-based

Blocks important negative signaling pathways, effective passive immunotherapy, and can be conjugated with drugs

Large and repeated doses needed for administrations, mainly functions with antigens on cell surface or circulating antigens, and short-life in vivo

DNA-based

Easy to design and produce, thermo stable, and activates cell-mediated responses

Induces weak humoral immune responses, requires electroporation or other devices for administration, and safety concern

Cell-based

Induces high level of specific immune response and can be individualized for patients

Passive immunotherapy, needs multiple administration, expensive production, and risk of contamination

Protein- and peptide-based vaccines

Cell-based vaccines

HBsAg and/or HBcAg or peptides derived from these viral proteins have been used. Although some trials showed effective induction of T cell responses and cytotoxicity in vitro, these responses are transient and not related to decrease in viral load or sero-conversion in HBeAg [23,24]. Proteinbased vaccines are also used with more potent adjuvants, and some vaccines are used in combination therapy with antiviral drug (e.g., lamivudine). However, results are inconsistent and controversial [25,26].

In a previous study [30], cytokine-induced killer (CIK) cells were used as cell-based vaccine. CIK cells comprise heterogeneous cell populations, which include effector cell population expressing both the T cell and NK cell markers (CD3+CD56+). CIK cells were shown to lyse target cells in a non-major histocompatibility (MHC)-restricted manner in tumors. In a clinical study, 21 CHB patients were transfused with autologous CIK cells for three successive infusions, and serum HBV DNA, HBeAg sero-conversion, and normalization of ALT were monitored. Administration of the CIK cells closely correlated with the decrease in serum HBV load and improvement in liver function in some patients [31]. DC pulsed cells were used for the treatment because DCs are dedicated antigen presenting cells, which are distributed throughout the body, and are important in antigen presentation to CD4+ and CD8+ cells. In a study involving 380 CHB patients, autologous DCs loaded with HBcAg18–27 peptide (FLPSDFFPSV) and HBV Pre-S2 44–53 peptide (SILSKTGDPV) were infused intravenously to patients nine times (up to 27 weeks) to evaluate the safety and efficacy of this treatment [32]. Among the 185 HBeAgpositive patients, 22.2% lost HBeAg and 16.2% seroconverted within 24 weeks. These values increased to 29.7% and 21.6%, respectively, at 48 weeks. Among the 195 HBeAg-negative-patients, 8.2% were HBsAg negative, and the HBsAg sero-conversion rate was 1.0% at 24 weeks. These values increased to 10.3% and 2.6% (5/195), respectively at 48 weeks. At 48 weeks, 25 of 185 (13.5%) HBeAg-positive patients presented a complete response, as defined by HBV DNA loss, ALT normalization, and HBeAg sero-conversion. By contrast, 105 of the 195 HBeAg-negative cases (53.9%) presented complete response. Therefore, this approach proved to be safe. In another study [33], intradermal HBsAg-pulsed DCs were administered one to three times in five CHB patients. This approach is also proved to be safe. In

DNA-based vaccines DNA-based vaccines could mimic the effect of live attenuated viral vaccines by eliciting cell-mediated immunity, in addition to inducing humoral responses, and thus are used as therapeutic vaccines for CHB. A DNA vaccine expressing HBV small (S) and middle (preS2 + S) envelope proteins was administered to 10 HBeAg-positive CHB patients. Proliferative responses in immune cells and IFN-γ-producing T cells specific for the preS2 or the S antigen were detectable in 50% to 100% of these patients, whereas transient seroconversion to anti-HBe were detected only in two patients [27]. DNA vaccine combined with antiviral drug treatment was also used. A pilot randomized controlled trial of HBV DNA vaccine enhanced by IL-2/IFN-γ fusion protein plasmid construct was administered to 39 CHB patients by electroporation under lamivudine chemotherapy. No data about the use of the DNA vaccine alone are available, but the HBeAg sero-conversion rate was only 9% both in the lamivudine + placebo and lamivudine + DNA vaccine groups [28]. Meanwhile, phase I/II trial in 70 CHB patients is being conducted to determine the effect of DNA vaccination treated with nucleotide analogs on T cell restoration and delayed viral reactivation after treatment discontinuation (www.ClinicalTrials.gov, Identifier: NCT00536627) [29].

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addition, antigen-specific humoral and cellular immune responses were detected in two and one patient, respectively. However, no therapeutic efficacy was reported. Antigen-antibody-based vaccines Appropriate proportion of antibody-antigen complex was originally used to immunize animals to produce higher titers of antibodies [34]. After 20 years, Celis et al. [35] showed that IgG subclass monoclonal antibodies against HBsAg complexed to the antigen considerably increased the amount of HBsAg captured and internalized by antigen-presenting cells, and the proliferation of mouse T cell clones, as well as the production of IFN-γ, also increased. Berzofsky [36] studied HIV peptide immunization in healthy volunteers and found that boosting with antigen-antibody complexes results in higher stimulation indices in T cell response compared with free antigen boosting. In 1994, we reported that solid-matrixDHBsAg-anti-DHBs complex could clear viremia and antigenemia in certain percentage of ducks persistently infected with duck HBV [37]. A pilot study on the use of HBsAg-HBIG (i.e., high-titer of immunoglobulin against HBsAg) complex was subsequently conducted to treat CHB patients [38]. The antigen-antibody-based therapeutic vaccine was formulated on the hypothesis that, by constructing a complex of HBsAg with anti-HBs, the tolerogen (HBsAg), antigen presenting cells (APCs) would be forced to uptake HBsAg via Fc receptors on the APCs of the tolerant host, thereby modulating the processing and the presentation of HBsAg to break immune tolerance versus HBV. To verify the therapeutic effects and mechanisms of the use of antigenantibody complexes as therapeutic vaccine, experimental studies have been conducted both in cells in vitro and in mice. Given that Fc receptors are critical in HBsAg uptake, when the Fc fragment of anti-HBs were digested by enzyme to delete the Fc fragment, but the Fabs were retained, remarkably reduced uptake of the complex by APCs was observed. In addition, given that the Fc receptors are also species specific, when goat anti-HBs were substituted for mouse anti-HBs to construct immunogenic complex (IC) with HBsAg, the uptake of the complex by mouse APC was drastically decreased, as well as the induction of immune responses in mice [39]. When HBsAg-anti-HBs (mouse antibodies) IC complex was used to immunize HBsAg positive transgenic mice, the decrease in the serum HBsAg, induction of anti-HBs, and specific cytolytic responses were all markedly higher than those in mice immunized with HBsAg or anti-HBs alone [40]. To study the mechanisms of IC in patients, DCs from CHB patients were incubated with HBsAg, anti-HBs, IC, or culture medium (control). Compared with the control group, DCs incubated with IC produced the highest number of MHC II molecules on DCs and IL-12 production. When the T cells from the same patients were incubated with their relevant DCs pre-incubated with IC, HBsAg, or anti-HBs, higher levels of IFN-γ and IL-2

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were observed from T cells which interacted with DCs in IC [41,42]. Phases I, IIA, and double-blind-placebo-controlled IIB, and III clinical trials of IC have been conducted after approval by the Chinese National FDA [41–43]. In summary, IC manufactured with HBsAg and HBIG has been proved to be safe for CHB patients. Among the treated patients, some underwent liver function flares during treatment, but all patients recovered subsequently with favorable therapeutic outcomes. In these trials, sero-conversion from HBeAg to anti-HBe was considered as the primary response, and decrease in viral load and normalization of liver functions were secondary responses. The HBeAg sero-conversion rate of CHB patients after six injections of IC (intramuscular injections of 60 µg of HBsAg in IC per injection every 4 weeks) and followed for six months is 21.8%, with 9% in the alum control group. However, when the number of immunization increased to 12 injections, HBeAg seroconversion rate of IC immunization decreased to 14.0%, whereas multiple injections of alum alone resulted in 21.9% HBeAg sero-conversion. Immune fatigue resulted in decreased cell-mediated immune response from overstimulation of IC, which could be the cause of the decreased response rate by 12 consecutive injections of IC [44]. This clinical trial experience showed that for vaccine therapy, appropriate dosage, time course, and immunization protocol should be carefully studied and designed. In addition, by optimizing the immunization protocol, another clinical trial with the use of IC to treat CHB patients is being conducted.

Perspectives on therapeutic vaccines for CHB CHB is considered a complex disease because of the diversity in the patients’ genetic backgrounds, clinical histories, and virological and immunological status. Therefore, in the future, therapeutic vaccination should be individualized, and new approaches are needed to improve efficacy. For protein- and peptide-based therapeutic vaccines, development of new adjuvants, particularly those that can effectively stimulate cell-mediated immune responses are recommended to improve their efficacies. Given that these therapeutic vaccines are easy to manufacture and easy to apply, their use in combination with agents to block inhibitory factors in patients may be beneficial. Antigen-antibody complex-based therapeutic vaccine can be considered as a novel form of antibody-mediated treatment. Traditionally, antibody-mediated treatment neutralizes the antigen in circulation, interacts with antigens expressed on cells or delivers drugs to the target cells. By contrast, ICs deliver antigen to immune cells with the use of its antibody, thereby resulting in the modulation of host immune responses and induction of more effective antigen presentation. This method can be explored further in treating other persistent diseases.

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The production of DNA therapeutic vaccines is inexpensive, and the products do not need cold chain transportation, which is an advantage for developing countries. Given that electroporation has been used for the administration of DNA vaccine; this approach markedly improves the delivery of DNA vaccines. Multivalent DNA vaccines containing different components need evaluation for the expression of all antigens in vivo and also for possible side-effects. Primeand-boost strategy has been shown to be effective in other vaccines and may also improve the efficacy of DNA vaccine. A triple complex therapeutic vaccine candidate containing HBsAg-anti-HBS complex and DNA from plasmid encoding the S gene of HBV as the adjuvant has excellent therapeutic effect in a transgenic mouse model [38]. The combination of exogenous antigen presentation pathway by IC and endogenous antigen presentation pathway by DNA is speculated to improve the effectiveness of the presentation and processing of HBsAg, which should be evaluated in clinical trials. For cell-based therapeutic vaccines, only CHB patients who are nonresponsive to immune stimulation should be administered with this treatment. Multiple infusions of autologous cells loaded with cytokines have been used to treat cancer patients. However, this approach is expensive and has risk of contamination, which may not be practical for treating a large number of patients. The reported effectiveness (50%) among HBeAg-negative is promising, and further studies are recommended to determine its effectiveness and mechanisms. We propose that the goals for developing therapeutic vaccination should consider the three “A”s, namely, affordable, available, and acceptable. We expect that therapeutic vaccination for CHB will be inexpensive and should be affordable for patients to use, available to treat patients in developing countries, as well as simple, effective, and acceptable. Immunotherapy is a method used to combat diseases, and its use in combination with antiviral drugs should be carefully designed to achieve synergetic efficacy.

Compliance with ethics guidelines Yumei Wen, Xuanyi Wang, Bin Wang, and Zhenhong Yuan declare that there is no conflict of interest. This manuscript is a review article and does not involve any research protocol requiring approval by the relevant institutional review board or ethics committee.

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