Recent Advances in HBV Vaccination

51
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 benecial 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 rst 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
RECENT ADVANCES IN HEPATITIS B
VACCINATION
Kazimierz Madalinski
Head, Laboratory of Immunology of Hepatotropic Viruses,
Department of Virology, National Institute of Public Health -
National Institute of Hygiene, Warsaw, Poland
Correspondence: Dr. Kazimierz Madalinski, Laboratory of Immunology of
Hepatotropic Viruses, Department of Virology, National Institute of Public Health,
National Institute of Hygiene, 24, Chocimska St, 00-791, Warsaw, Poland.
E-mail: kmadalinski@pzh.gov.pl
DOI: 10.4103/0972-9747.58805
Author proofs done******
REVIEW ARTICLE
[Downloaded free from http://www.hepatitisbannual.org on Saturday, July 24, 2010, IP: 119.154.58.159]
52
raises many hopes and with sufcient 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
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.
Epidemiology
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:
Insufcient sterilisation of multiple-use equipment (endoscopes,
catheters, etc.);
Insufcient 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.
1
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
53
subcontinent, about 5% of persons are subject to chronic infection.
The worst situation is observed in the developing world (most of
the Asia-Pacic region and above all, sub-Saharan Africa) where
the rate of infection spans between 8 and 10%.
2
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.
3
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.
HBV genotypes
Eight different HBV genotypes (A-H) have been identied,
based on sequence genome analysis. Most of them show distinct
geographical distribution.
4
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 inuence
of different genotypes was found on the activity and progress
of liver disease, seroconversion rate to anti-HBe, and treatment
54
efcacy. Spontaneous seroconversion to anti-HBe was observed
earlier in persons infected with B than with C genotype. Treatment
effectiveness (IFN-) was higher in patients with genotype A than
B and D.
1
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.
4-6
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.
Anti-HBV vaccines
Currently, the following recombinant anti-HBV vaccines are
produced and available in the market [Tables 1a and b].
Experimental recombinant vaccines (Table 1b, positions 1
Table 1a: Characteristics of the currently used, commercial
second-generation vaccines; product name, producer, doses
used
Name Producer Doses
Engerix B Glaxo Smith Kline, 10 and 20 g
Rixensart, Belgium
Gen-H-B-Vax Merck Sharp Dohme, USA 2.5, 5 and 10 g
H-B-Vax II Merck Sharp Dohme, USA 5, 10, 40 g
H-B-Vax PRO Merck Sharp Dohme, USA 5, 10, 40 g
GenHevac B Pasteur Merieux 20 g
Recombivax Merck Sharp Dohme 2.5, 5 g pediatric
10 g adults
40 g dialysis,
leukemia etc.
Hepavax-Gene Green Cross Vaccine, Korea 10 and 20 g
Euvax B Ikson Factory LG Chem, Korea 10 and 20 g
Table 1b: Third generation vaccines containing surface antigen
S, with pre-S1, pre-S2 peptides
Bio-Hep-B (Sci-B-Vac)* Biotechnology General, Rehovot, Israel
Hepa Gene 3 Exogene / Hexal Biotech Germany
Hepacare Medeva Gt. Britain
Hepimmune* Berna Biotech Switzerland
*Vaccines of the same origin
55
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.
7,8

More than half of the immunized children showed the appearance
of anti-preS1 and/or anti-preS2 antibodies in the circulation.
9
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.
10
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 g doses
of vaccine in 50% of the newborns.
11
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).
12,13
Immunization
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.
14,15
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 Childrens 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.
16
These remarkable achievements were obtained
56
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 sufcient 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 rst year after
immunization, starting from the top values of antibodies, relatively the
biggest decline of antibodies was obtained. One of the rst 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.
17
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 rst year, there was
a very steady decline of antibodies between 4th - ~7th year, since
57
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.
18- 21
The level of persisting antibodies and rate
of responders with protective antibodies in a given time hardly
reected 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.
21
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:
(1) The level of lymphocyte proliferation to HBsAg parallels the
concentration of serum anti-HBs antibodies;
(2) Positive lymphocyte proliferation to HBsAg may appear together
with borderline anti-HBs ( 10 IU/L), or in the absence of
antibodies.
22,23

It was concluded that cellular immunity, represented by
specic 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 specic immunization carry the potential to respond to
HBV challenge bibliography position.
18
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
58
are not needed in healthy responders to HBV vaccine.
12,13,22,23
Unresponsiveness
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 insufciency (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 difcult 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 identied in immunized children worldwide.
24
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 specic immunoglobulin (HBIG). This is
the main cause of the appearance of the escape mutants; liver
transplant recipients also develop the same after HBIG prophylaxis.
25

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.
26
A mathematical model of HBV transmission was
described, to investigate the potential pattern for emergence of
escape mutants; the authors discussed the vaccine modication,
with a wider epitope range.
27
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
59
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.
21
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, Mikoajewicz
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.
28
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% efcacy). The study was performed on a very big group
of 719 persons.
29
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.
30,31
Approximately 20 yrs after the launch of the Expanded
Program of Immunization, the stepwise decrease of HCC cases was
observed: rst 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.
32,33
60
DNA-based vaccines
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-specic 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.
34
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.
35
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 rst tested in animals, mice, monkeys
and pigs, and the results were promising. The rst 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.
34,35
In particular, induction of T CD4+
and CD8+ response and protective antibody response in human
volunteers was observed in the study mentioned above.
34

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.
36
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;
61
alone, or in combination with nucleoside analog, lamivudine.
37
The
study has shown that specic 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.
38
This has raised hopes that using similar technology to incorporate
preS-S genes of HBV into edible plants will enable us to prot
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.
39
The following genetically modied
plants were tried in these procedures: lettuce, carrot, potato, cherry
tomato, lupin, maize and tobacco.
40,41
Researchers are also considering introducing the banana as
a perfect delivery system.
42
It is interesting to note that many good
experimental studies in this eld were performed in such countries
as Poland, USA, Egypt, India, China and USA.
43
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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Source of Support: Grant PBZ-KBN 119/P05/05 from the National
Ministry of Science and High Education, Poland, Conict of Interest: None
declared.

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HBsAg DNA vaccine consists of microscopic gold

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