| Abstract|| |
Hepatitis B virus is a microorganism formed in the excess of surface antigen which is devoid of nucleic acid. Surface antigen of HBV was from the beginning the natural candidate for the vaccine which was thus produced by isolation of plasma HBsAg and later substituted by recombinant protein(s).
The Extended Program of Immunization was beneficial for the reduction of HBV incidence in the populations of many participating countries. It is further postulated that HCC incidence in the world was also reduced at least in the portion caused by hepatitis B virus. Persistence of anti-HBV immunity was first measured by quantitative anti-HBs assay determined at 1 month post vaccination cycle, and then at different time points, even up to 12-15 years. The frontier of 10 IU/L (mIU/ml) is a mark of sustained immunity. However, cellular immunity studies revealed that this kind of response is very important in the defense against the virus and may last longer than the detectable antibodies. It was shown that 'full' surface vaccines, i.e. preS+S, may give stronger immunity and are good even for neonates. The next generation vaccines are DNA-based and plant-based HBV vaccines. This last category raises many hopes and with sufficient immunogenicity could ensure the most comfortable route of administration.
Keywords: Anti-HBs, DNA vaccines, edible vaccines, Hepatitis B virus (HBV), hepatitis B surface antigen (HBsAg), immunization, immunogenicity, pre-S1 antigen, vaccine
|How to cite this article:|
Madalinski K. Recent advances in Hepatitis B vaccination. Hep B Annual 2008;5:51-65
| Introduction|| |
It is estimated that ca. 350 million people (~5% of the world population) live with chronic Hepatitis B virus (HBV) infection, which may progress to cirrhosis and/or hepatocellular carcinoma (HCC). One million new cases with HB infection appear annually. The death rate from cirrhosis and HCC is estimated at 500,000 persons/year. Vaccination against hepatitis B becomes a powerful tool to combat the spread of the disease.
In European countries present routes of HBV transmission differ, depending on the age group. Young people (15-25 years) acquire the infection most commonly from heterosexual contacts (especially with multiple partners). The second cause in this group is drug addiction by intravenous route, using infected needles, syringes etc. Altogether, up to 40% drug addicts are infected with HBV. On the other extreme are persons >60 yrs of age, whose HBV infections, connected in ~90% with medical procedures, should be treated as nosocomial. These infections appear usually after hospital treatment and are the result of:
Epidemiology of HBV has very uneven spread in the world. Infection is least common in Northern and Western Europe and North America (less than 1% people are chronically infected). A little higher rate of infection, between 1-1.5% was noted in Central and South Eastern Europe. In the Middle East and the Indian subcontinent, about 5% of persons are subject to chronic infection. The worst situation is observed in the developing world (most of the Asia-Pacific region and above all, sub-Saharan Africa) where the rate of infection spans between 8 and 10%. 
- Insufficient sterilisation of multiple-use equipment (endoscopes, catheters, etc.);
- Insufficient hygiene of the personnel, esp. not to frequent washing of hands, no change of gloves even after small medical procedures.
- The other routes of infection, especially in adult population, may be hair-dressing and cosmetic procedures. 
Characteristics of HBV and pathogenesis
Hepatitis B virus belongs to Hepadnaviridae family. The full virion (Dane particle), 42 mm in diameter, contains nucleocapsid and surface proteins. Circular, partly double stranded DNA composes the HBV genome. The long strand (L-) contains 3000-3300 base pairs (bp) and short strand (S+) contains 1700-2899 bp. The negative strand is composed of four open reading frames: pre-S/S, pre-C/C, P and X. Region pre-S/S contains 3 starting codons coding the surface proteins: (1) large protein (L), a product of pre-S1, pre-S2 and S genes, (2) middle protein (M), a product of pre-S2 and S genes, (3) small protein (S), a product of S gene and a main component of surface antigen, HBsAg.
Region pre-C/C contains two starting codons coding nucleocapsid antigens: (1) HBeAg, a product of pre-C gene, and (2) HBcAg, a product of C gene.
Region P codes polymerase of three enzymes activity: (1) DNA-dependent DNA polymerase, (2) reverse transcriptase, and (3) ribonuclease H. Region X codes regulatory protein HBx, of transactivating properties. HBx protein increases expression of HLA class I molecules, prerequisite of the proper presentation of HBV peptides to CD8+ cytotoxic lymphocytes; it can also modulate the other host and viral genes expression. 
Products of pre-S/S region, i.e. surface proteins of the HBV, are the main components of the protective vaccine. This is composed usually of S-protein exclusively, or of large protein, i.e. pre-S1 and pre-S2 proteins together with S protein.
Eight different HBV genotypes (A-H) have been identified, based on sequence genome analysis. Most of them show distinct geographical distribution.  In Europe, over 80% of isolates represent genotype A, the rest -- genotype D; while genotypes B and C are prevalent in Asian carriers/ chronic hepatitis B patients. The influence of different genotypes was found on the activity and progress of liver disease, seroconversion rate to anti-HBe, and treatment efficacy. Spontaneous seroconversion to anti-HBe was observed earlier in persons infected with B than with C genotype. Treatment effectiveness (IFN-α) was higher in patients with genotype A than B and D.  However, another observation suggests that genotype A predominates in patients with chronic active (aggressive) hepatitis, while genotype D can be found more commonly in patients with acute self-resolving hepatitis. ,, As the distribution of HBV genotypes differs around the globe, it correlates in certain countries with the origin of immigrants and overall structure of migration.
Currently, the following recombinant anti-HBV vaccines are produced and available in the market ([Table 1]a and b].
Experimental recombinant vaccines ([Table 1]b, positions 1 and 4) were produced via the expression of pre-S1, pre-S2 and S-protein components of HBV, e.g. in Chinese hamster ovary (CHO) cells. These vaccines were evaluated for safety, tolerability and immunogenicity in healthy adults and children.
The 3 rd generation preS/S vaccines produced in CHO cells have revealed excellent immunogenicity in humans and the rapid onset of the antibody response to the S-component of the vaccine. , More than half of the immunized children showed the appearance of anti-preS1 and/or anti-preS2 antibodies in the circulation.  In addition, immunization with these kinds of vaccines gives early onset of antibodies and may require only two injections to obtain an excellent antibody response.  High titers of anti-HBs antibodies in newborns were elicited after only two injections; antibodies towards pre-S1 and/or pre-S2 antigens were synthesized after 5 mg doses of vaccine in 50% of the newborns.  It has been suggested that a high titer of anti-HBs may ensure a longer duration of both the humoral and cellular immunity (see below). ,
An Extended Program of Immunization (EPI), including hepatitis B vaccine for children just after birth (neonates) has been launched in over 150 countries by the World Health Organization. , Fortunately, in addition to HBV vaccine dispatched from the governmental resources and coordinated by WHO, the vaccine for children is available through the assistance of Global Alliance for Vaccines and Immunization and Global Funds for Children's Vaccines. The address for further information is:
www.who.int/health topics/hepatitis B.
Subsequent studies revealed that mass hepatitis B immunization was effective in preventing HBV infection and resulted in a decrease in the occurrence of HCC in children and adults living in countries where hepatitis B is endemic. For example, of 22 European countries which entered the EPI program and could be evaluated around the year 2004 - in most countries with a high initial incidence of hepatitis B, a substantial decrease of the number of cases during the 12-yr period was noted. The most spectacular decrease was observed in Poland (incidence: from ~40/100.000 to 4.5/100.000 population), then in Bulgaria and Romania (35 to 15), Lithuania (20 to 10), Czech Republic and Slovakia (10 to 5), respectively.  These remarkable achievements were obtained with the plasma-derived vaccine, and later with the yeast-derived, recombinant vaccines against HBV infection.
After proper immunization with recombinant, S-containing vaccine, adults would respond in ~97% and children in 98.5% of cases. The synthesis of anti-HBs antibodies at a protective level: ≥10 IU/L and ≥100 IU/L (cut-off values for normal and immunocompromised persons, respectively) and sufficient cellular immunity should be achieved. The geometric mean titer would oscillate around 25,000 IU/L in adults and ca. 35,000 IU/L in children, as measured within 1 month from the last vaccine dose. It was interesting to know how long the post-vaccination immunity lasts. Very early observations indicated that within the first year after immunization, starting from the top values of antibodies, relatively the biggest decline of antibodies was obtained. One of the first studies describing the evolution of the anti-HBs antibodies concentration after full-cycle immunization, i.e. 0/1/6 month dose schedule, of adults (n = 280) with HBV vaccine was made by Gesemann and Scheiermann.  As mentioned before, the starting point was within 1 month after the 3rd vaccine dose; then measurements were performed every year. The following results were obtained:
t = 1; F = 37844
t = 12; F = 4541
t = 18; F = 3034
t = 25; F = 1875
t = 37; F = 1449
t = 49; F = 991
- - - - - - - - - - - - - - - -
t = 82; F = 855
where t = months after the end of vaccination; F = concentration of antibodies in IU/L. Geometric mean titer (GMT) of antibodies was counted for each time point.
The vaccinated persons were healthy young people; thus the level of antibodies at 1 month post immunization was really high. Taking the concentration of anti-HBs at this time point (37,844 IU/L) as 100%, we can calculate that ca. 12% of antibodies remained after 12 months; 8.9% remained after 18 months, and 2.6% after 49 months (4 years). After fast decline within first year, there was a very steady decline of antibodies between 4th - ~7th year, since at 82 months 2.04% of antibodies still remained. The decrease of the antibody concentration from 1 to 82 months can be illustrated by the logarithmic curve (data not shown).
In other studies, different population groups like Alaskan natives, Iranian, Taiwanese and Chinese children and Italian adults were evaluated for persistent anti-HBs antibodies after vaccination. After a mean period ranging from 10 to 15 years, ca. 50% (at 15 yrs) or 75-85% (at 10-14 yrs) remained with the GMT antibody level of &≥10 IU/L. ,,, The level of persisting antibodies and rate of responders with protective antibodies in a given time hardly reflected any geographic and/or ethnographic differences, being roughly the same.
A certain level of humoral immunity, after 4th dose - booster given post 5.6 years from initial vaccination of health care workers, can persist up to 18 years.  The practical question, how long protective anti-HBV immunity persists, was solved by the next set of experiments, exploring the cellular immunity (i.e. lymphocyte proliferation) of vaccinated vs. 'naive' subjects. Control antigens for lymphocyte proliferation were tetanus/diphtheria and the target antigen was recombinant HBsAg. These experiments revealed:
It was concluded that cellular immunity, represented by specific lymphocyte proliferation persists longer than the protective antibodies after successful immunization. This conclusion goes in parallel with observations from pediatrics that neonates and infants up to 1 year have a predominance of naive (75%) over memory (25%) lymphocytes in their circulation. At the age >10 yrs the reverse is true; i.e. memory (CD4/CD45+RO) cells predominate naive cells (CD4/CD45+RA) in the circulation. Memory cells raised during the specific immunization carry the potential to respond to HBV challenge bibliography position. 
- The level of lymphocyte proliferation to HBsAg parallels the concentration of serum anti-HBs antibodies;
- Positive lymphocyte proliferation to HBsAg may appear together with borderline anti-HBs (≤10 IU/L), or in the absence of antibodies. ,
On this basis, a consensus view was formulated that after successful immunization of a child or an adult against HBV, the protective immunity lasts at least 15 yrs. Thus, booster injections are not needed in healthy responders to HBV vaccine. ,,,
Another problem connected with the anti-HBV vaccination is the status of the immune system of the vaccinated person, i.e. the problem of unresponsiveness. The relatively high unresponsive state is connected with situations and diseases, such as neonates of HBsAg+ mothers, children or adults after renal transplants, with nephritic syndrome and end-stage renal insufficiency (4-15% nonresponders; NR). The highest rate of nonresponsiveness represents patients with leukemia, lymphoma and/or solid tumors (30% NR). To overcome the low response rate of these patients, a full 4-dose scheme is usually applied (0/1/2/6 months; 0/1/2/12/ months) and the dose of the vaccine could be doubled. This helps to obtain better results in many cases, even when using the 'conventional' vaccine.
The use of third generation vaccine, i.e. containing all surface proteins pre-S1, pre-S2 and S should be an almost ideal solution to immunize such difficult groups of children and adults.
To summarize, remarkable results were already achieved in prevention of hepatitis B infection. However, mutants of the 'a' determinant of HBsAg, capable of escaping vaccination, have been identified in immunized children worldwide.  The principal mechanism of appearance of 'escape mutants', usually at 145 th position of the S protein within 'a' determinant, is described for infants. Neonates born to HBV infected mothers receive HBsAg vaccine and hepatitis B specific immunoglobulin (HBIG). This is the main cause of the appearance of the escape mutants; liver transplant recipients also develop the same after HBIG prophylaxis.  Universal vaccination in many countries of the world has accelerated the trend for the appearance of HBsAg 'a' determinant mutations with amino acid changes critical for immune escape in vaccinated children, who become carriers through vertical or horizontal transmission.  A mathematical model of HBV transmission was described, to investigate the potential pattern for emergence of escape mutants; the authors discussed the vaccine modification, with a wider epitope range.  Furthermore, it was suggested that the 'ideal vaccine' should mimic the immunological response developed during the natural infection. Also, there are views that inclusion of pre-S proteins within recombinant HBV vaccine might induce an adequate antibody response that would prevent the infectivity of HBV escape mutants. Another important observation was that at a mean of 12 years after health care workers' immunization with two types of vaccine, the response was 2.3x higher to plasma-derived than to yeast-derived vaccine.  Plasma-derived vaccine apparently might contain a certain amount of pre-S proteins in addition to S.
It cannot be clearly stated that all high-risk groups with impaired immunity were immunized with pre-S+ S containing vaccines and the response was systematically compared with the response to S-containing vaccine. There were, however, observations on the potential of BioHep B to overcome unresponsiveness to S-containing vaccines in several adults and children (Madalinski K, Gregorek H, Woynarowski M, Mikolajewicz J; unpublished data). Similar results were obtained in a study of 925 health care workers, previous nonresponders to commercial vaccines, of whom 75% responded successfully after one dose of 'Hepacare' triple antigen (preS+ S) vaccine.  The problem of nonresponsiveness to conventional recombinant S vaccine was taken up again by a European group. This is especially interesting, since European health authorities recommend the use of a cut-off of 100 IU/L (instead of ≥10 IU/L) of anti-HBs antibodies for seroprotection in persons at increased risk; i.e. patients with chronic diseases and health care workers. The authors have found that the third generation pre-S/S hepatitis B vaccine was much better in overcoming nonresponse than the conventional vaccine (>80 vs. 50% efficacy). The study was performed on a very big group of 719 persons.  Thus, there are many possibilities at present to combat, at least partially, the present unresponsiveness to conventional HBV vaccines.
Among persons who become chronically infected with HBV during childhood, ca. 25% will run the risk of death from HBV-related cirrhosis or cancer. Well over 80% of hepatocellular carcinoma (HCC) cases are induced both by HBV and HCV; this neoplasm takes 5th place among all cancers. ,
Approximately 20 yrs after the launch of the Expanded Program of Immunization, the stepwise decrease of HCC cases was observed: first in South-East Asia, but also in other regions. The role of HBV in inducing liver cancer has substantially decreased, but in favor of hepatitis C virus. ,
These are the next generation preparations. It was shown in laboratory animals that DNA vaccination appeared an effective method to induce protective immunity against pathogenic antigens, including HBV proteins. Studies showed DNA vaccination as an inducer of HBV-specific immune response in strains of mice not responding to immunization with HBV proteins.
DNA vaccines also showed the potential to induce T cell responses, including CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T helper cells with Th1 type cytokines.  HBsAg-DNA can be introduced into the host by intramuscular or intradermal route using a needle with syringe. An alternative is using gene-gun, Biolistic®, PowderJectTM, Accell® , or particle-mediated DNA delivery. DNA-coated microscopic gold particles are delivered by a needle-free device directly into the epidermic cells.  For example, PowderJect® HBsAg DNA vaccine consists of microscopic gold beads coated with a plasmic expression vector, which contains an eukaryotic expression cassette encoding the entire 226-aa. HBsAg protein from HBV of subtype 'adw'. Transcription of the coding region (HBsAg) is regulated by the human CMV IE1 enhancer/promoter, intron A sequence components and the growth hormone polyadenylation signal.
DNA vaccines were first tested in animals, mice, monkeys and pigs, and the results were promising. The first human studies were performed between 1999-2001, showing very good safety, and no local or systemic side effects, except for little or no pain at the site of vaccination. , In particular, induction of T CD4+ and CD8+ response and protective antibody response in human volunteers was observed in the study mentioned above. 
The next step was to prove the overcoming of the nonresponse or very low response to conventional HBV vaccine in human subjects who received HBV DNA vaccine by particle-mediated epidermal delivery.  Breaking of nonresponse was successful in >50% subjects; while all subjects with waning antibody levels after conventional vaccines responded successfully, in some instances the response was long-lasting.
Another approach, taken up on an animal model (duck hepatitis B) was to use DNA-based vaccine in therapeutic trials; alone, or in combination with nucleoside analog, lamivudine.  The study has shown that specific DNA immunization to HBV structural proteins was able to induce complete virus clearance (in about 23% of animals). Combination therapy of DNA vaccine given together with lamivudine increased the rate of elimination to 38%, as a sustained response. Thus, the combination therapy using DNA vaccine and lamivudine represents an interesting approach for chronic hepatitis B therapy.
Plant-based HBV vaccines
The idea to provide easy delivery of vaccine to adults but especially children directed researchers to the not-so-easy procedure of producing plant-based viral vaccines. Indeed, upon expression in yeasts, recombinant HBV surface proteins have been used from several years for parenteral 'syringe' immunization.  This has raised hopes that using similar technology to incorporate preS-S genes of HBV into edible plants will enable us to profit from the enormous potential of gut-associated lymphoid tissue (GALT) of laboratory animals, as well as humans, for the synthesis of humoral and cellular immunity against HBV. The big problem remains immunogenicity of plant-incorporated HBV proteins, thus cholera toxin was added as mucosal adjuvant in order to obtain maximum antibody response.  The following genetically modified plants were tried in these procedures: lettuce, carrot, potato, cherry tomato, lupin, maize and tobacco. ,
Researchers are also considering introducing the banana as a perfect delivery system.  It is interesting to note that many good experimental studies in this field were performed in such countries as Poland, USA, Egypt, India, China and USA. 
| Acknowledgment|| |
This work was supported by grant PBZ-KBN 119/P05/05 from the National Ministry of Science and High Education, Poland.
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Laboratory of Immunology of Hepatotropic Viruses, Department of Virology, National Institute of Public Health, National Institute of Hygiene, 24, Chocimska St, 00-791, Warsaw