Hepatitis B Annual

Current Issue  

Back Issues   


Search Login    Users online: 150 Print this page  Email this page Small font sizeDefault font sizeIncrease font size 
>>> Ahead of Print <<<

REVIEW ARTICLE Table of Contents   
Year : 2005  |  Volume : 2  |  Issue : 1  |  Page : 31-55
Preventive and therapeutic approaches against hepatitis B virus by vaccine

Third Department of Internal Medicine, Ehime University School of Medicine, Toon city, Ehime 791-0295, Japan

Click here for correspondence address and email


Hepatitis B virus (HBV) infection is caused by a small enveloped DNA virus that infects the liver. Approximately 2 billion people of the world have been infected by HBV and 300-350 million of them are chronically infected with the virus. The annual global mortality rate from HBV infection and its sequelae is 1-2 million people. Control of HBV infection is a major challenge of contemporary virology, hepatology, immunology, and vaccinology. There are two important aspects of this problem. The first is the prevention of new HBV infection. Transmission of HBV from HBV-infected persons to uninfected healthy individuals can be blocked by interrupting the transmission cycle of the virus. But, this is extremely hard and time-consuming, especially in view of the socio-economic status and health care delivery system of most of the developing nations, where HBV carrier rate is high. Another practical solution to this problem is by immunoprophylaxis using vaccines against hepatitis B. Commercially available hepatitis B vaccines induce protective antibody against HBV in more than 90% of apparently healthy individuals. However, these vaccines are ineffective in about 5-10% of normal individuals, most immunosuppressed subjects and also in liver transplanted patients with HBV background. Accordingly, a more potent prophylactic vaccine against HBV is needed. The concept of using vaccine for treatment of chronic diseases (vaccine therapy) has existed for quite a long time, but very few clinical trials with vaccine therapy have been conducted to assess their efficacy and clinical usage. During the last decade, some studies have reported the use of hepatitis B vaccine to treat patients with chronic hepatitis B. The present regimen of vaccine therapy for patients with chronic hepatitis B is safe, but is not very effective as an independent therapeutic tool. This article reviews the development of more potent prophylactic vaccines against HBV and better regimens of therapeutic vaccines for patients with chronic hepatitis B with special reference to the recent developments in the relevant fields.

How to cite this article:
Akbar S, Onji M. Preventive and therapeutic approaches against hepatitis B virus by vaccine. Hep B Annual 2005;2:31-55

How to cite this URL:
Akbar S, Onji M. Preventive and therapeutic approaches against hepatitis B virus by vaccine. Hep B Annual [serial online] 2005 [cited 2023 Dec 5];2:31-55. Available from: https://www.hepatitisbannual.org/text.asp?2005/2/1/31/29374

   Main Text Top

Why are more potent prophylactic vaccines against HBV infection needed?

The major limitation for developing vaccine against hepatitis B virus (HBV) is the absence of technology to grow HBV in vitro . Moreover, there is no suitable animal model that allows random replication of HBV. Hepatitis B vaccine was developed from the plasma of HBV-infected persons. The purification and inactivation of both HBV and contaminated agents in human blood were done earlier by pepsin digestion, urea dissociation-resuscitation, and treatment with formaldehyde.[1] With the advancement of molecular technology, plasma-derived vaccine was replaced by yeast-derived vaccine in 1986.[2] Both plasma-derived hepatitis B vaccine and yeast-derived hepatitis B vaccine contain small surface protein of HBV and administration of these vaccines induces antibody to hepatitis B surface antigen (anti-HBs) in more than 90% HBV-uninfected, healthy normal individuals.[3] However, these vaccines are unable to induce protective levels of anti-HBs (10 mIU/ml) in about 5-10% of apparently healthy subjects.[4] Moreover, commercially-available hepatitis B vaccines are not effective in inducing anti-HBs in a considerable proportion of immunosuppressed individuals such as patients infected with human-immune deficiency virus[5] or patients receiving immunosuppressant drugs.[6] In addition, drug users also exhibit low or non response to hepatitis B vaccines.[7] Finally, administration of hepatitis B vaccine does not induce anti-HBs in liver transplanted patients when the donor or recipients are infected with HBV.[8] This is an extremely important issue in the context of liver transplantation. Liver transplantation is the treatment of choice for end-stage liver diseases and this surgical procedure is getting popular day by day. It is now possible to perform liver transplantation from living donor; however, this life-saving approach offers little practical benefit if the donors or recipients are infected with HBV. This is especially important in some countries of Far-East Asia where about 70% people show either previous or present infection with HBV.[9] In order to protect transplanted livers, these patients are given hepatitis B immunoglobulin (HBIG) on a regular basis to prevent reactivation of HBV for the rest of their life.[10] Unfortunately, HBIG is extremely costly, and this has considerably restricted liver transplantation in these subjects.

Generally 10 mg of HBsAg are used in commercial hepatitis B vaccines. It has been postulated that more powerful prophylactic vaccines against HBV may be prepared by increasing the amount of HBsAg or by adding other HBV-related proteins in commercially-available hepatitis B vaccines. Accordingly, two proteins related to the surface antigen of HBV (pre S2 and S1 antigens) have been added in hepatitis B vaccines.[11],[12] However, most trials have revealed that immunization with vaccines containing multiple proteins of HBV or increased amounts of HBV-related antigens provide very little, if any, added benefits.[13] In addition to this, other vaccines against HBV such as DNA-based vaccine and cytotoxic T cell (CTL)-inducing vaccine have been reported. However, more pilot studies will be required before using them in humans.

Concept of immune therapy against chronic HBV infection

Worldwide, there are approximately 300-400 million individuals who are chronically infected with HBV.[14] It is not completely clear why some HBV-infected patients clear the virus after an acute infection, whereas the others are unable to do so and become chronic HBV-carriers. Circumstantial evidences suggest that the age of infection and the immune status of the hosts are important factors in this regard. HBV infection in adult life is often clinically inapparent and most of the acutely infected adults recover spontaneously. These persons either clear or control the virus after acute infection. Only a small proportion (5-10%) of HBV-infected adults become chronic HBV carriers. By contrast, up to 90% of children who acquire the virus from HBV-infected mothers, become chronically infected with HBV.[15],[16] Patients chronically infected with HBV exhibit various different types of liver diseases. In some patients, the disease is progressive in nature and a considerable proportion of these eventually develop clinical features of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.[17]

Patients with HBV-related chronic liver diseases have been treated with various types of drugs in different parts of the world. In Western countries, drugs with antiviral potential have been widely used in these patients during the last two decades. Now it is evident that eradication of HBV from a chronic HBV carrier by an anti-viral drug is an unachievable goal. Therapy of chronic HBV carriers is now aimed at reducing HBV replication, improvement of liver function tests and induction of seroconversion from HBeAg to anti-HBe.[18] Currently, in Western countries, the two approved therapies for chronic hepatitis B with clinical beneficial effect are alpha-interferon and lamivudine. Alpha-interferon therapy, which combines antiviral and immunostimulant properties, results in suppression of HBV replication in only some patients with chronic hepatitis B.[19] Lamivudine, a nucleoside analogue, leads to a rapid discontinuation of HBV replication as well as to a frequent improvement of the necroinflammatory activity of liver diseases and to a lesser extent of fibrosis.[20] However, short-term treatment with lamivudine leads to a frequent relapse of HBV replication. On the other hand, long-term treatment (to avoid the risk of relapse) has been shown to result in virological breakthrough related to the selection of resistant viral variants with an annual incidence of 15-25%.[21] Recently, adefovir is also being used either alone or in combination with lamivudine to treat patients with chronic hepatitis B. However, renal toxicity of adefovir may be a limiting factor. Significant virological and clinical improvements are seen in only some, but not all patients who are given both drugs together.[22] Moreover, treatment with these antiviral agents results in seroconversion to anti-HBe in limited patients with chronic HBV infection.

All these limitations of antiviral therapy in chronic HBV carriers indicate that development of a more potent therapeutic regimen is badly needed for these patients; however the strategy for developing this is not clear. In order to have a proper perspective for this, there is a need to review our understanding of the pathogenesis of chronic HBV carrier state as well as that of acute resolving hepatitis.

Pathogenesis of chronic HBV carrier state

HBV is a non cytopathic virus and the magnitude of HBV replication does not correlate with the degree of liver injury in chronic HBV carriers. Patients with chronic HBV infection exhibit HBV-specific immune responses which are weak, transient, narrowly focused and antigenically-restricted.[23] This is quite different from the nature of immune responses of HBV-infected individuals who clear the virus after an acute infection.[24],[25] It has been presumed that HBV-specific CD8+ CTL is responsible for effective control of HBV because these cells are capable of recognizing and destroying HBV-infected hepatocytes. However, it is now evident that destruction of HBV-infected hepatocytes by HBV-specific CTL may represent only one of the mechanisms of HBV clearance from chronic HBV carriers. Interestingly, patients with chronic hepatitis B exhibit massive recruitment of non-antigen specific mononuclear cells in the liver. The role of these mononuclear cells in the clearance of HBV and in the induction of damage of the liver cells is not well understood.[26] Various cells of the immune systems such as CD8+ HBV-specific CTL, CD8+ HBV-non specific T lymphocytes, CD4+ helper T cells and possibly macrophages and natural killer cells may have dominant roles in both viral clearance and liver cell damage in chronic HBV carriers. The most important finding regarding immune pathogenesis of chronic HBV carrier state has been elucidated by the findings that clearance of the HBV from infected hepatocytes probably does not require massive lysis of hepatocytes, but could be mediated by secretion of type 1 cytokines (type-1 and II interferons and tumor necrosis factor alpha) secreted by cells of the innate and adaptive immune systems.[27] Moreover, a role of CD8+ CTL in clearance of HBV from the host has now been down played because there has been no correlation between the frequencies of CD8+ T cells and hepatocytes lysis.[28] Thus, probably the mechanisms that control HBV replication might also cause damage of the hepatocytes. This information is very important for designing new therapeutic regimen for chronic HBV infection.

One of the characteristic features of chronic HBV carriers is their impaired immune response to HBV-related antigens. Patients with chronic HBV infection are usually immune competent. They respond to various microbial agents properly. Vaccination against various infectious agents also induces protective immunity in these subjects. However, they are unable to induce adequate levels of HBV-specific immune responses in vitro and vivo . Accordingly, it is postulated that if proper HBV-specific immune responses can be induced in chronic HBV carriers, they may have therapeutic potential. The important question is what types of immune responses should be induced for therapeutic purposes? The immune therapy for chronic HBV carriers must maintain a critical balance. Typically, this therapy should induce immune response to HBV-related antigens, reduce HBV replication and normalize alanine transamitransferase (ALT). On the other hand, the outcome of the immune therapy should be optimum so that there is no massive damage to the hepatocytes. This can not be achieved by administration of cytokines and polyclonal immune modulators because some studies regarding this has shown only limited benefit. On the other hand, use of polyclonal immune modulators may induce more severe degree of liver damage in these patients.

Vaccine therapy in chronic HBV carriers

Dienstag et al first used vaccine containing HBsAg to assess the production of anti-HBs in chronic HBV carriers.[30] This was not termed as 'therapeutic vaccine' or 'vaccine therapy' because the aim of the study was to assess if chronic HBV carriers can produce anti-HBs following injection with the prophylactic vaccine. The conclusions of this trial were negative because none of the patients exhibited detectable levels of anti-HBs in the sera. However, interestingly 8 of 16 patients showed decreased levels of ALT and one of them seroconverted to anti-HBe.

Pol et al were the first to report the therapeutic efficacy of a prophylactic hepatitis B vaccine in patients with chronic hepatitis B in 1994, and the therapy was then named 'vaccine therapy'.[31] They administered three standard vaccine doses containing HBsAg and pre-S2 antigen in 32 patients with chronic hepatitis B. Efficacy of this therapy was defined as a sustained loss of HBV DNA or a decrease of more than 50% of HBV DNA compared to prevaccination titers. Vaccine therapy resulted in dissapearance of serum HBV DNA in 12 of 32 patients, 3 months after the end of 3 monthly vaccination. Responders to vaccine therapy developed anti-HBe in the sera. Vaccine therapy was uneventful except for a transient rise in serum ALT in some patients which subsequently normalized.

During the last decade, several clinical trials of vaccine therapy have been reported in patients with chronic HBV infection, some of which are controlled trials, whereas, the others are pilot studies. Wen et al have shown that efficacy of vaccine therapy can be enhanced if vaccines containing HBsAg/anti-HBs complex is administered to chronic HBV carriers.[32] Although, the investigators did not study the mechanism of action underlying better therapeutic potential of antigen/antibody complex hepatitis B vaccine, they postulated that better activation of antigen-presenting cells may be responsible for this.

A controlled trial of vaccine therapy was reported in 2001 in which a total of 118 patients who never received any previous anti-HBV therapy were enrolled in the study. These patients were given 5 intramuscular injections with vaccine containing 20 g of a preS2/S vaccine. The evaluation was done 12 months after therapy commencement. The rate of HBV DNA negativation was significantly higher in vaccinated group compared to control group (16.3% versus 2.7%) at 3 months after the end of vaccination.[33]

In the above-mentioned studies, patients with chronic hepatitis B were administered hepatitis B vaccine 3 to 6 times. We conducted a pilot study of vaccine therapy in 11 patients with chronic hepatitis B in which vaccine containing 20 g of HBsAg was administered once every 2 weeks 12 times. Eight patients showed reduction of HBV DNA and normalization of serum ALT. Moreover, the rate of seroconversion to anti-HBe was higher in vaccinated group compared to the control group.[34] Other investigators have also reported about vaccine therapy in patients with chronic hepatitis B in which they have used either hepatitis B vaccine alone or other immune modulators combined with hepatitis B vaccine. Ren et al too have documented antiviral potentiality of vaccine therapy in chronic HBV carriers.[35] Senturk et al reported that therapeutic vaccine containing preS2 is efficient to induce HBV DNA negativation in 24% and decreased HBV DNA in 40% of patients with chronic hepatitis B.[36] However, Dikici et al did not find any notable therapeutic efficacy of vaccine therapy in pediatric patients in the immunotolerant phase of chronic HBV infection.[37]

Vaccine therapy conducted in patients with chronic hepatitis B from Europe and Japan have reported reduced replication of HBV, negativation of HBV, normalization of ALT and seroconversion to anti-HBe in varying proportion of patients with chronic hepatitis B, but none of the patients seroconverted to anti-HBs. However, Yalcin et al from Turkey first reported that 3 of 31 patients cleared HBsAg and developed anti-HBs due to monthly vaccination with a preS2/S vaccine for 3 times.[38]

Vaccine therapy for liver transplanted subjects with a HBV background

In addition to its use as a therapeutic vaccine for chronic HBV carriers, hepatitis B vaccine has also been used to block de novo replication of HBV in liver transplanted patients, in which either the donor or recipient were infected with HBV. The idea was to induce anti-HBs by hepatitis B vaccines in these subjects after live transplantation; anti-HBs would then neutralize HBV in these live transplanted subjects. This will eventually reduce the usage of costly HBIG. There are conflicting reports regarding the efficacy of prophylactic hepatitis B vaccine in liver transplanted subjects with HBV background.[39]

Vaccine therapy with peptide vaccine in different types of adjuvant

As the therapeutic outcome of HBsAg-based vaccine is inconclusive in patients with chronic hepatitis B, several approaches were undertaken to increase the immune modulatory capacity of hepatitis B vaccine in patients with chronic hepatitis B. Some investigators have used peptide epitopes that are recognize by CTL as immunogens for the development of therapeutic vaccines. Vitiello et al covalently attached two additional components: a T helper peptide epitope of HBV and two lipid molecules to produce a therapeutic vaccine. Although this vaccine induced HBV-specific CTL, clinical efficacy of this vaccine has not been documented in patients with chronic hepatitis B.[40]

With the understanding that an effective therapeutic vaccine should induce both humoral and cellular immune responses, alternative adjuvant containing MF59 was used in patients with chronic HBV infection. Preliminary data in abstract form claimed that 11 of 13 patients developed anti-HBs to such a vaccine.[41] Unfortunately, no further data was published regarding the therapeutic efficacy of this vaccine.

Vaccine therapy by DNA vaccine encoding HBV-related antigens

DNA vaccine encoding HBV-related antigens have been widely tested in animal models of HBV carrier state, usually in HBV transgenic mouse (HBV-Tg). DNA vaccines have potent anti-HBs induction capacity in HBV-Tg. A single injection of DNA vaccine encoding HBV-related proteins induced HBV-specific immune responses in several lines of HBV-Tg.[42],[43]

Recently, DNA vaccine containing HBV-related antigens have been used in human volunteers. DNA vaccine encoding HBsAg has been shown to induce anti-HBs in nonresponders to HB vaccination.[44] DNA vaccination did not show any side effects in these subjects. Data of clinical trial of DNA vaccine encoding HBV-related antigens in patients with chronic hepatitis B are yet to be published.

The importance of HBsAg-based vaccine for vaccine therapy

There is considerable data regarding the limitation and scope of different types of vaccine such as antigen-based vaccine, peptide-based vaccine and DNA vaccine in patients with chronic hepatitis B. Peptide-based vaccine and DNA vaccine have shown potent therapeutic efficacy in some selective studies, but it is uncertain whether these vaccines will ever be used for therapeutic purposes in clinics. It will be hard to get permission from the ethical committees of most of the countries for using DNA vaccine against HBV infection. The long-term adverse effects of DNA-based therapy constitute major limitation of these therapeutic agents.

On the other hand, there are some advantages of HBsAg-based vaccines for treating patients with chronic hepatitis B. It should be explained why HBsAg-based vaccines were used for vaccine therapy, although there are many antigens of HBV such as hepatitis B core antigen, hepatitis B e antigen and hepatitis B X antigen. Therapeutic vaccines have been used for other pathological conditions too mainly in patients with cancers. There is a fundamental difference in immune therapy vis-a-vis cancer subjects and patients with chronic hepatitis B. Immune therapy in patients with cancers is done to destroy cancer cells. On the contrary, destruction of HBV-infected hepatocytes is not the main purpose of immune therapy in patients with chronic hepatitis B. Rather, the aim is to minimize the destruction of HBV-infected hepatocytes because almost all hepatocytes are infected with HBV in these patients. The purpose of vaccine therapy is to induce HBsAg-specific immune responses which were lost during chronic HBV infection. If the lost HBV-specific immune responses can be regained, the immune competent host may tackle HBV by their own immune response capabilities Thus, the purpose of vaccine therapy is not to induce HBV-specific cytotoxic T cells (CTL), rather to induce HBV-specific CD4+ T cells and subsequent HBsAg-specific B cells responses. The use of HBsAg for vaccine therapy has been influenced by various factors. HBsAg is the antigen that induces protective immunity against HBV, and this antigen is commercially-available in human consumable form too. However, use of hepatitis B core antigen-based or other HBV antigen-based vaccines in animal models of chronic HBV carrier state should be encouraged in order to develop insights regarding their possible clinical usage in future. In addition, some studies can also be carried out with vaccines containing a mixture of HBsAg and other HBV-related antigens in animal models of HBV carrier state.

Why is there a lack of general acceptance of vaccine therapy for treating chronic HBV carriers?

HBsAg-based therapeutic vaccine is not only safe for patients with chronic HBV carriers, it is also cheap compared to antiviral therapies for these patients, and the duration of therapy is not so long. However, there are some unresolved questions regarding the efficacy of this therapy. Although, various clinical trials have shown that vaccine therapy is some how effective in some patients with chronic HBV infection, it is true that much work needs to be done to get an optimum or most effective protocol of vaccine therapy. Besides very few clinicians are actually using this therapy for their patients. This indicates that there is a lack of general acceptance of vaccine therapy in practice. In fact, a state of confusion is prevailing regarding the real therapeutic efficacy of vaccine therapy in chronic HBV carriers and probably for any type of immune therapy in various clinical situations. Hepatologists and immunologists are equally responsible for this state of uncertainty in clinical application. Vaccine therapy has not been conducted in a systematically way in patients with chronic hepatitis B. Some investigators have shown that vaccine therapy is effective for treating chronic HBV carriers, whereas, others did not find considerable therapeutic effect of vaccine therapy in their patients. The main causes underlying this diversity regarding therapeutic efficacy of vaccine therapy in chronic HBV carriers are as follows: (1) The virological and immunological status of the patients in different studies are completely different. The efficacy of vaccine therapy is dependent on the nature of the hosts. Vaccine therapy trials have been conducted without any proper stringent inclusion or exclusion criteria, although it is clear that different factors relating to the hosts and the viruses influence the clinical outcome of therapy of patients with chronic hepatitis B. (2) The therapeutic regimen of vaccine therapy has not been optimized. Some studies have used only 3 vaccinations where, as others have administered 12 doses of vaccination. While the vaccine has been administered once a month by some, others have vaccinated once every 2 weeks. (3) There are no proper standardised criteria for evaluation of the clinical efficacy of vaccine therapy.

There is a need to have further insights into the virological and immunological status of the patients who might benefit from vaccine therapy. The characteristics of the patients are very important for the efficacy of antiviral therapy. Antiviral therapy is not effective in immune tolerant phase of chronic HBV carriers, and vaccine therapy seems to be ineffective in this phase of HBV infection.

Different types of therapeutic regimens are used for vaccine therapy in chronic HBV carriers. The nature of the vaccine is different too. In some studies, vaccines containing HBsAg have been used, whereas, vaccines containing HBsAg and preS2 have been used in others. Besides, therapeutic vaccines have been administered for a variable schedule of 3-12 times, and there is no controlled study addressing the dose schedule. It is still unknown whether a single regimen of vaccine therapy is optimum or vaccine therapy should be conducted once in every 6 months or 12 months.

The criteria of assessment of efficacy of vaccine therapy are the most confusing part of these different studies on vaccine therapy. Complete HBV negativation, 50% reduction of HBV DNA, seroconversion to anti-HBe, and improvement of liver damages have been used to assess the efficacy of vaccine therapy. Among the criteria which are now being used for evaluation of therapeutic efficacy of vaccine therapy, complete negativation of HBV DNA in the sera from chronic HBV carriers is not an achievable goal and should be omitted as an evaluation criterion. HBV DNA persists in all persons infected with HBV even if they are cured completely after an acute hepatitis. Although dissappearance of HBsAg and production of anti-HBs in chronic HBV carriers due to vaccine therapy have been reported by Yalcin et al ,[38] this seems to be a very special situation because other studies could not document such drastic efficacy of vaccine therapy.

We critically analyzed why a state of confusion was prevailing about vaccine therapy in patients with chronic hepatitis B. The major problem lies in our perception about the scope and limitation of the different therapies. At the outset, we have to understand that vaccine therapy is not a drug. It has not emerged from the brain of multinational drug companies or from their research and development branches. As a result, there has been no financial and technical support for conducting clinical trials in the different phases of vaccine therapy. This therapy has been proposed and practised by clinicians and immunologists, and hence the different facets of this therapy should be optimized by scientists and clinicians. The birth of vaccine therapy is an excellent development in the field of medicine, and the unbearable burden of labor pain accompanying its birth must also be borne by the scientists.

Role of antigen-presenting dendritic cell (DC) in cancellation of immune response defects against so-called tolerogenic antigen

There is a need to develop more powerful regimens of therapeutic vaccine for chronic HBV carriers. This will ultimately contribute to development of more powerful prophylactic vaccine against HBV. In order to achieve this goal, we reviewed (1) the mechanism underlying the production of anti-HBs in normal individuals by hepatitis B vaccine and (2) the scientific basis of vaccine therapy in chronic HBV carriers. HBsAg is a T cell-dependent antigen. Accordingly, purposeful and interactive dialogue among T cells, B cells and antigen-presenting cells is essential for production of anti-HBs in vivo . In general, after administration of vaccine, HBsAg in the vaccine is recognized and internalized by antigen-presenting DCs at the site of injection. After processing HBsAg in their endosomal compartments, DCs migrate to the regional lymphoid tissues to interact with clonally selected T and B lymphocytes at the so-called immunological synapses.[45],[46],[47] HBsAg-specific B lymphocytes and plasma cells are produced and secrete anti-HBs which is detected in the sera.

It has been described for decades that the chronic HBV carriers are unable to produce anti-HBs or HBsAg-specific T cells mainly due to immunogenic tolerance that arise from interaction of the hosts with HBV during neonatal period at which time the immune system is comparatively immature. This view was proposed by immunological investigations and received overwhelming support from hepatologists because most of the neonates who were transmitted by HBV-infected mothers became chronic HBV carriers. The original theory of neonatal tolerance was put forth by Burnett et al[48] but that was never proved by experimental evidences. In the mid 1990s, Matzinger et al showed that exposure of neonates with antigen does not induce neonatal tolerance; rather neonates are immune competent and are able to induce immune responses to various types of microbial agents.[49] In the context of chronic HBV infection too, our laboratory clearly showed that the concept of neonatal tolerance should be reviewed.[50]

In fact, induction of antigen-specific immune responses at all ages of human and higher animals are dependent on processing and presentation of antigen by DCs. In order to clarify the role of DCs in chronic HBV infection we decided to study the function of DCs in HBV carriers. As it was not possible to isolate DCs from human in the early 1990s, we used HBV-Tg, as an animal model of HBV carrier state. HBV-Tg expressed HBsAg from 17th day of gestation. Study of HBV-Tg revealed that these mice resembled human HBV carriers regarding their expression and response to HBsAg.[51] A series of studies in our laboratory revealed that the inability of HBV-Tg to exhibit anti-HBs and HBsAg-specific T cells was due to the impaired functions of the DCs of HBV-Tg.[52] This led us to activate the DCs of HBV-Tg in an antigen-specific manner by administration of vaccine containing HBsAg (vaccine therapy). Study on the mechanism of action of vaccine therapy in HBV-Tg revealed that circulating HBsAg was not inducing antigen-specific immune response in HBV-Tg because HBsAg particles were not regarded as dangerous or harmful entity in HBV-Tg. However, injection of vaccine containing complete Freund's adjuvant or aluminum hydroxide possibly provided the requisite danger signals so that DCs of HBV-Tg recognized the injected HBsAg and induced HBsAg-specific immune responses. The efficacy of vaccine therapy was confirmed by conducting four placebo-controlled trials of vaccine therapy in HBV-Tg, each of which was done for 12 months.[53],[54],[55],[56] We also received an opportunity to acquire better insights into the role of DCs in vaccine therapy. Vaccine therapy induced anti-HBs in about 40% of HBV-Tg (vaccine responders), but not in the rest 60% HBV-Tg (vaccine non responders). A critical role of DCs in the therapeutic efficacy of vaccine therapy was evident from the observation that DCs from vaccine responder HBV-Tg had significantly better antigen-presenting capacity compared to DCs from vaccine non responders HBV-Tg.[57]

Next generation prophylaxis and therapeutic vaccine for prevention of HBV infection and for treatment of chronic HBV carriers: Study in animal model of chronic HBV infection.

In order to develop more potent therapeutic vaccines, we loaded HBsAg on DCs in vitro . This was done to minimize the influence of endogenous DCs of HBV-Tg on the therapeutic efficacy of vaccine therapy in HBV-Tg because DCs of HBV-Tg had low antigen-presenting capacities. HBsAg-pulsed DCs had significantly higher immune modulatory capacity compared to unpulsed DCs. HBV-Tg were administered with 2 injections of HBsAg-pulsed DCs at an interval of 2 weeks. Serial observation revealed that all HBV-Tg injected with HBsAg-pulsed DCs developed anti-HBs in their sera within 6 weeks of second immunization.[58]

HBsAg-pulsed DCs were also efficient in inducing anti-HBs in immunosuppressed HBV-Tg. A group of immunosuppressed HBV-Tg was prepared by daily administration of tacrolimus in HBV-Tg. Spleen DCs were isolated from these immunosuppressed HBV-Tg and loaded with HBsAg in vitro . Immunosuppressed HBV-Tg was administered with HBsAg-pulsed DCs from immunosuppressed HBV-Tg. Only two injections of immunosuppressed DCs from immunosuppressed HBV-Tg induced anti-HBs in immunosuppressed HBV-Tg.[59],[60]

HBsAg-pulsed DC vaccine for human

Elegant data regarding the therapeutic efficacy of antigen-pulsed DCs were reported in animal models of various human diseases. Moreover, antigen-pulsed DCs were also used for treating patients with cancers. But, it was an open question whether antigen-pulsed DCs would ever be used in immune competent patients for induction of antigen-specific immune response. The major concern was patient's safety, because these DCs may induce antigen-specific immunity in an uncontrollable manner that may lead to induction of autoimmunity. The next issue was about the methodology of production of antigen-pulsed human DCs. In mouse, antigen-pulsed DCs have been prepared from bone marrow DCs or splenic DCs, but not from blood DCs. In humans, only blood DCs can be used for this purpose.

In this context, we decided to produce HBsAg-pulsed human DCs and planned a clinical trial of antigen-pulsed DCs in human volunteers. It was possible to prepare HBsAg-pulsed human blood DCs by culturing monocyte-derived human blood DCs with commercially available hepatitis B vaccine. A single injection of HBsAg-pulsed DCs in normal volunteers either sharply increased the serum levels of anti-HBs in anti-HBs-positive volunteers or induced anti-HBs in anti-HBs negative normal volunteers. All volunteers were meticulously checked for any signs or symptoms suggestive of generalized inflammatory conditions, liver and kidney function tests and tests for autoimmunity.[61] No adverse effects were documented in any volunteer during a period of 12 months after administration of HBsAg-pulsed DCs. We have also produced HBsAg-pulsed DCs from patients with chronic hepatitis B in vitro and are planning to study their safety and efficacy in patients with chronic hepatitis B.

   Conclusions Top

Prevention of HBV infection: Prophylactic vaccine

Hepatitis B vaccine is a major success in the history of vaccinology. All children and high-risk population of the world should be vaccinated with this vaccine. This vaccine has now been included in 'Expanded Program of Immunization' in most of the countries of the world, especially in the developing nations of Asia and Africa. This will definitely curtail the transmission of HBV to a certain degree. Reports from different countries have shown that vaccination with commercially available vaccines have drastically reduce new HB infection as well as the prevalence of chronic HBV carriers in some countries who adopted childhood immunization with hepatitis B vaccines in the early 1980s. As the commercially-available vaccines have some limitations, we have documented that HBsAg-pulsed DC vaccine may represent a more powerful prophylactic vaccine against HBV when adequate protection against HBV can not be attained by conventional vaccines. HBsAg-pulsed DC vaccine can be used in vaccine non responders, immunosuppressed individuals and patients with liver transplantation. Indeed, much work needs to be done to evolve the best protocol for producing HBsAg-pulsed human blood DCs. Studies should target and focus on the numbers of DCs, the amount of HBsAg, the duration of pulsing and the route of administration for preparing more immunogenic HBsAg-pulsed DCs. The concept to develop more potent prophylactic vaccine against HBV has been provided. HBsAg-pulsed human blood DCs have been prepared and clinical trials have been conducted. The next step is to reproduce our data regarding anti-HBs producing capacity of HBsAg-pulsed DCs among different races and in different types of individuals.

Treatment of chronic HBV infection: Development of more potent therapeutic vaccines

Vaccine therapy using commercially available hepatitis B vaccine has been conducted in some countries of the world to treat patients with chronic hepatitis B. The present regimen of vaccine therapy is safe for patients with chronic hepatitis B. It is also cheaper compared to antiviral therapies, and the duration of therapy is less compared to anti viral therapies. However, there is some controversy about the efficacy of vaccine therapy in chronic hepatitis B. In some studies, a moderate to good therapeutic potential of this therapy have been reported. On the other hand, no significant beneficial effects of this therapy were found in other reports. These data about therapeutic efficacy have mainly been influenced by several factors such as (1) the nature of the patients studied, (2) the regimen of the vaccine therapy, and (3) the criteria of evaluation of therapeutic efficacy of vaccine therapy.

The real position of vaccine therapy deserves critical appraisal in view of the different therapeutic choices for chronic hepatitis B. 300-350 million people of the world are chronically infected with HBV and at least 20% of them deserve active therapy for their diseases. Type 1 interferon has been used in these patients for the last 2 decades, mainly in the developed countries. Although, this drug is exhorbitantly costly, many chronic HBV carriers in the developing countries have been given a single course of type 1 interferon at the expense of their whole asset. Very few patients with chronic hepatitis B have got long-term benefit from this treatment. Lamivudine is now being used in different countries of the world for treating chronic hepatitis B. The formidable barrier of this therapy is emergence of resistant strain and breakthrough hepatitis in a considerable proportion of patients. Although, the health care delivery system of the developed countries can handle lamivudine-resistant cases, it is almost impossible to handle these cases in most of the countries of Asia and Africa, where most of the chronic HBV carriers live. It is a reality that many hepatologists have been telling their patients that the available drugs are not effective for your problems and there is no option but to accept the unbearable. Many of these patients are waiting to see the final days of their life in which they will have to accommodate with liver cirrhosis, hepatocellular carcinoma and liver failure; all of which will compromise the quality of their life to a great extent. What can be done for these patients? Vaccine therapy seems a promising alternative therapeutic choice. The most dominant positive side of vaccine therapy is its safety. We propose that controlled trials of vaccine therapy should be conducted in different countries of the world with proper inclusion and exclusion criteria of the patients. More studies need to be conducted about the optimum doses of vaccine and duration of therapy. Next, a combination of antiviral therapy and vaccine therapy may be a good therapeutic choice for chronic hepatitis B. We have recently reported that the efficacy of lamivudine therapy can be increased and the side effects can be decreased by administrating vaccine for 12 times during lamivudine therapy.[62] Indeed, many models for combination therapy can be generated for patients with chronic hepatitis B. Finally, HBsAg-pulsed DC vaccine will represent a next-generation immune therapy for chronic hepatitis B. However, over expectation from vaccine therapy will negatively affect its clinical usage. We must realize the concept of modern market economy in the context of vaccine therapy for patients with chronic hepatitis B. Drugs developed by commercial companies (type 1 interferon and lamivudine) and special immune therapy originated by scientists (vaccine therapy) should not be compared using similar criteria. Hopefully, data pertaining to vaccine therapy in chronic hepatitis B inspires enough optimism so that lessons learnt at the benches can really be utilized for the patients' benefit. It is the duty of the clinicians to optimize the protocol of vaccine therapy in view of the need of their patients.

   References Top

1.Buynak EB, Roehm RR, Tytell AA, Bertland AU 2nd, Lampson GP, Hilleman MR. Vaccine against human hepatitis B. J Am Med Assoc 1976;235:2832-4.  Back to cited text no. 1  [PUBMED]  
2.Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 1982;298:347-50.  Back to cited text no. 2  [PUBMED]  
3.Nossal GJ. The global alliance for vaccines and immunization: A millennial challenge. Nat Immunol 2000;1:5-8.   Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Desombere I, Willems A, Leroux-Roels G. Response to hepatitis B vaccine: Multiple HLA genes are involved. Tissue Antigens 1998;51:593-604.  Back to cited text no. 4  [PUBMED]  
5.Bruguera M, Creades M, Salinas R, Costa J, Grau M, Sans J. Impaired response to recombinant hepatitis B vaccine in HIV-infected persons. J Clin Gastroenterol 1992;14:27-30.  Back to cited text no. 5    
6.Idilman R, Colantoni A, De Maria N, Ustun C, Sam R, Ing TS, et al . Impaired antibody response rates after high dose short interval hepatitis B virus vaccination of immunosuppressed individuals. Hepatogastroenterology 2003;50:217-21  Back to cited text no. 6    
7.Rumi M, Colombo M, Romeo R, Boschini A, Zanetti A, Gringeri A, et al . Suboptimal response to hepatitis B vaccine in drug users. Arch Intern Med 1991;151:574-8.  Back to cited text no. 7    
8.Angelico M, Di Paolo D, Trinito MO, Petrolati A, Araco A, Zazza S, et al . Failure of a reinforced triple course of hepatitis B vaccination in patients transplanted for HBV-related cirrhosis. Hepatology 2002;35:176-81.  Back to cited text no. 8    
9.Lok AS. Hepatitis B infection: Pathogenesis and management. J Hepatol 2000;32:89-97.  Back to cited text no. 9    
10.Nymann T, Shokouh-Amiri MH, Vera SR, Riely CA, Alloway RR, Gaber AO. Prevention of hepatitis B recurrence with indefinite hepatitis B immune globulin (HBIG) prophylaxis after liver transplantation. Clin Transplant 1996;10:663-7.  Back to cited text no. 10  [PUBMED]  
11.Rendi-Wagner P, Wiedermann G, Stemberger H, Kollaritsch H. New vaccination strategies for low- and non-responders to hepatitis B vaccine. Wien Klin Wochenschr 2002;114:175-80.  Back to cited text no. 11  [PUBMED]  
12.Kim MJ, Nafziger AN, Harro CD, Keyserling HL, Ramsey KM, Drusano GL, et al. Revaccination of healthy nonresponders with hepatitis B vaccine and prediction of seroprotection response. Vaccine 2003;21:1174-9.   Back to cited text no. 12    
13.Singh M, O'Hagan DT. Recent advances in vaccine adjuvants. Pharm Res 2002;19:715-28.  Back to cited text no. 13    
14.Rizzetto M, Zanetti AR. Progress in the prevention and control of viral hepatitis B: Closing remarks. J Med Virol 2002;67:463-6.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.Lok AS. Hepatitis B infection: Pathogenesis and management. J Hepatol 2000;32:89-97.  Back to cited text no. 15    
16.Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Ann Rev Immunol 1995;13:29-60.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Rehermann B. Immune responses in hepatitis virus infection. Semin Liver Dis 2003;23:21-38.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.Lok AS, Heathcorte EJ, Hoofnagle JH. Management of hepatitis B: 2000 - summary of a workshop. Gastroenterology 2001;120:1828-53.  Back to cited text no. 18    
19.Hilleman MR. Overview of the pathogenesis, prophylaxis and therapies of viral hepatitis B, with focus on reduction to practical applications. Vaccine 2001;19:1837-48.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]
20.Liaw YF, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, et al . Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. Gastroenterology 2000;119:172-80.  Back to cited text no. 20    
21.Schalm SW, Heathcote J, Cianciara J, Farrell G, Sherman M, Willems B, et al . Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: A randomized trial. Gut 2000;46:562-8.  Back to cited text no. 21    
22.Nguyen MH, Wright TL. Therapeutic advances in the management of hepatitis B and hepatitis C. Curr Opin Infect Dis 2001;14:593-601.  Back to cited text no. 22  [PUBMED]  [FULLTEXT]
23.Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, et al . The role of virus-specific CD8+ cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269-80.  Back to cited text no. 23    
24.Rehermann B. Immune responses to hepatitis B virus infection. Semin Liver Dis 2003;23:21-38.  Back to cited text no. 24    
25.Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest 1997;99:1472-7.  Back to cited text no. 25    
26.Chisari FV. Hepatitis B virus transgenic mice: Models of viral immunobiology and pathogenesis. Curr Top Microbiol Immunol 1996;206:149-73.  Back to cited text no. 26    
27.Gallimore A, Dunnrese T, Hengartnet H, Zinkerangel RM, Rammensee HG. Protective immunity does not correlate with the hierarchy of virus-specific cytotoxic T lymphocytes responses to naturally processed peptides. J Exp Med 1998;187:1647-57.   Back to cited text no. 27    
28.Rehermann B, Ferrari C, Pasquinelli C, Chisari FV. The hepatitis B virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response. Nat Med 1996;2:1104-8.  Back to cited text no. 28    
29.Conjeevaram HS, Lok AS. Management of chronic hepatitis B. J Hepatol 2003;38:S90-103.  Back to cited text no. 29    
30.Dienstag JL, Stevens CE, Bhan AK, Szmuness W. Hepatitis B vaccine administered to chronic carriers of hepatitis b surface antigen. Ann Intern Med 1982;96:575-9.  Back to cited text no. 30    
31.Pol S, Driss F, Michel ML, Nalpas B, Berthelot P, Brechot C. Specific vaccine therapy in chronic hepatitis B infection. Lancet 1994;344:342.  Back to cited text no. 31    
32.Wen YM, Wu XH, Hu DC, Zhang QP, Guo SQ. Hepatitis B vaccine and anti-HBs complex as approach for vaccine therapy. Lancet 1995;345:1575-6.  Back to cited text no. 32    
33.Pol S, Nalpas B, Driss F, Michel ML, Tiollais P, Denis J, et al . Efficacy and limitations of a: Specific immunotherapy in chronic hepatitis B. J Hepatol 2001;34:917-21.  Back to cited text no. 33    
34.Horiike N, Akbar SM, Ninomiya T, Abe M, Michitaka K, Onji M. Activation and maturation of antigen-presenting dendritic cells during vaccine therapy in patients with chronic hepatitis due to hepatitis B virus. Hepatol Res 2002;23:38-47.  Back to cited text no. 34    
35.Ren F, Hino K, Yamaguchi Y, Funatsuki K, Hayashi A, Ishiko H, et al . Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection. J Med Virol 2003;71:376-84.  Back to cited text no. 35    
36.Senturk H, Tabak F, Akdogan M, Erdem L, Mert A, Ozaras R, et al . Therapeutic vaccination in chronic hepatitis B. J Gastroenterol Hepatol 2002;17:72-6.  Back to cited text no. 36    
37.Dikici B, Kalayci AG, Ozgenc F, Bosnak M, Davutoglu M, Ece A, et al . Therapeutic vaccination in the immunotolerant phase of children with chronic hepatitis B infection. Pediatr Infect Dis J 2003;22:345-9.  Back to cited text no. 37    
38.Yalcin K, Acar M, Degertekin H. Specific hepatitis B vaccine therapy in inactive HBsAg carriers: A randomized controlled trial. Infection 2003;31:221-5.  Back to cited text no. 38    
39.Sanchez-Fueyo A, Rimola A, Grande L, Costa J, Mas A, Navasa M, et al . Hepatitis B immunoglobulin discontinuation followed by hepatitis B virus vaccination: A new strategy in the prophylaxis of hepatitis B virus recurrence after liver transplantation. Hepatology 2000,31:496-501.  Back to cited text no. 39    
40.Vitiello A, Ishioka G, Grey HM, Rose R, Farness P, LaFond R, et al . Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans. J Clin Invest 1995;95:341-9.  Back to cited text no. 40    
41.Heineman TC, Clements-Mann ML, Poland GA, Jacobson RM, Izu AE, Sakamoto D, et al . A randomized, controlled study in adults of the immunogenicity of a novel hepatitis B vaccine containing MF59 adjuvant. Vaccine 1999;17:2769-78.  Back to cited text no. 41    
42.Thermet A, Rollier C, Zoulim F, Trepo C, Cova L. Progress in DNA vaccine for prophylaxis and therapy of hepatitis B. Vaccine 2003;21:659-62.  Back to cited text no. 42    
43.Oka Y, Akbar SM, Horiike N, Joko K, Onji M. Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice. Immunology 2001;103:90-7.  Back to cited text no. 43    
44.Rottinghaus ST, Poland GA, Jacobson RM, Barr LJ, Roy MJ. Hepatitis B DNA vaccine induces protective antibody responses in human non-responders to conventional vaccination. Vaccine 2003;21:4604-8.  Back to cited text no. 44    
45.Mellman I, Steinman RM. Dendritic cells: Specialized and regulated antigen processing machines. Cell 2001;106:255-8.   Back to cited text no. 45    
46.Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245-52.  Back to cited text no. 46    
47.Onji M. Dendritic cells in clinics. Springer: Tokyo; 2004.  Back to cited text no. 47    
48.Burnet FM, Stone JD, Edey M. The failure of antibody production in the chick embryo. Aust J Exp Biol Med Sci 1950;28:291-7.  Back to cited text no. 48    
49.Matzinger P. Tolerance, danger and extended family. Ann Rev Immunol 1994;12:991-1045.  Back to cited text no. 49    
50.Kurose K, Akbar SM, Yamamoto K, Onji M. Production of antibody to hepatitis B surface antigen (anti-HBs) by murine hepatitis B virus carriers: Neonatal tolerance vs antigen presentation by dendritic cells. Immunology 1997;92:494-500.  Back to cited text no. 50    
51.Araki K, Miyazaki J, Hino O, Tomita N, Chisaka O, Matsubara K, et al . Expression and replication of hepatitis B virus genome in transgenic mice. Proc Natl Acad Sci USA 1989;86:207-11.  Back to cited text no. 51    
52.Akbar SM, Onji M, Inaba K, Yamamura KI, Ohta Y. Low responsiveness of hepatitis B virus transgenic mice in antibody response to T-cell-dependent antigen: Defect in antigen presenting activity of dendritic cells. Immunology 1993;78:468-75.  Back to cited text no. 52    
53.Akbar SM, Kajino K, Tanimoto K, Michitaka K, Horiike N, Onji M. Placebo-controlled trials of vaccination with hepatitis B virus surface antigen in hepatitis B virus transgenic mice. J Hepatol 1997;26:131-7.   Back to cited text no. 53    
54.Akbar SM, Yamamoto K, Abe M, Ninomiya T, Tanimoto K, Masumoto T, et al . Potent synergistic effect of sho-saiko-to (TJ-9), a herbal medicine during vaccine therapy in a murine model of hepatitis B virus (HBV)-carrier. Eur J Clin Invest 1999;29:786-92.  Back to cited text no. 54    
55.Akbar SM, Abe M, Masumoto T, Horiike N, Onji M. Mechanism of action of vaccine therapy in murine hepatitis B virus carriers: Vaccine-induced activation of antigen presenting dendritic cells. J Hepatol 1999;30:755-64.  Back to cited text no. 55    
56.Akbar SM, Horiike N, Onji M. Prognostic importance of antigen presenting dendritic cells during vaccine therapy in murine hepatitis B virus carriers. Immunology 1999;96:98-108.  Back to cited text no. 56    
57.Akbar SM, Furukawa S, Horiike N, Onji M. Vaccine therapy for hepatitis B virus carrier. Curr Drug Targets Infect Disord 2004;4:93-101.  Back to cited text no. 57    
58.Akbar SM, Furukawa S, Hasebe A, Horiike N, Michitaka K, Onji M. Production and efficacy of a dendritic cell-based therapeutic vaccine for murine chronic hepatitis B virus carrier. Int J Mol Med 2004;14:295-9   Back to cited text no. 58    
59.Furukawa S, Akbar SM, Hasebe A, Horiike, N, Onji M. Induction and maintenance of anti-HBs in immunosuppressed murine hepatitis B virus carrier by a novel vaccination approach: Implication during liver transplantation in hepatitis B virus-infected subjects. J Gastroenterol 2004;39:851-8.  Back to cited text no. 59    
60.Furukawa S, Akbar SM, Hasebe A, Horiike, Onji M. Production of hepatitis B surface antigen-pulsed dendritic cells from immunosuppressed murine hepatitis B virus carrier:evaluation of immunogenicity of antigen-pulsed dendritic cells in vivo . Immunobiology 2004;209:551-7.  Back to cited text no. 60    
61.Fazle Akbar SM, Furukawa S, Onji M, Murata Y, Niya T, Kanno S, et al . Safety and efficacy of hepatitis B surface antigen-pulsed dendritic cells in human volunteers. Hepatol Res 2004;29:136-41.   Back to cited text no. 61    
62.Horiike N, Akbar SM, Michitaka K, Joukou K, Yamamoto K, Kojima N, et al . In vivo immunization by vaccine therapy following virus suppression by lamivudine: A novel approach for treating patients with chronic hepatitis B. J Clin Virol 2005;32:156-61.  Back to cited text no. 62    

Correspondence Address:
Morikazu Onji
Third Department of Internal Medicine, Ehime University School of Medicine, TO ON city, Ehime 791-0295
Login to access the Email id

Source of Support: None, Conflict of Interest: None

Rights and PermissionsRights and Permissions


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
    Email Alert *
    Add to My List *
* Registration required (free)  

    Main Text

 Article Access Statistics
    PDF Downloaded693    
    Comments [Add]    

Recommend this journal