W O R L D H E A L T H ~~{~~~ ORGANISATION MONDIALE ORGANIZATION ..
DE LA SANTE
SECOND INTERCOUNTRY SCIENTIFIC GROUP MEETING ON LIVER DISEASES
Damascus, 4-8 February 1984
E M / ~ N D . I N C . S ~ . L I V . D I S / ~
1 6 J a n u a r y 1984
Agenda i t e m 5.
SPECIFIC SCREENING TECHNIQUES FOR
MARKERS OF HEPATITIS B INFECTION
D r K . Nishioka* WHO Consu l t an t
* Vice P r e s i d e n t , Tokyo M e t r o p o l i t a n I n s t i t u t e of Medical S c i e n c e , Tokyo
and D i r e c t o r , WHO C o l l a b o r a t i n g C e n t r e f o r Reference and Research on V i r a l H e p a t i t i s
After first discovery of Australia antigen and its direct relationship
withPosttransfusion hepatitis by Agar gel diffusion technique by Okochi and
Murakami, serological methods for detection of HBV markers and corresponding
antibodies with higher sensitivity and specificity have been developed in our
country. These are immune adherence hemagglutination (IAHA) for detection of
HBsAg (Mayumi, 1971), Reversed passive hemagglutination (RPHA) for HBsAg (Juji,
1969), modified passive hemogglutination (PHA) for HBsAb (Imai, 1971), IAHA
for antiHBc antibody (Tsuda, 1975), PHA for HBe antigen and antibody (Takahashi,
1977), immune adherence for HBs antigen antibody complex (Tsuda, 1979, Takai,
1981), RIA using anti-human IgM coated beads for IgM anti HBc (Oguro, 1981),
high speed liquid chromatography PHA for IgM anti HBs (Naito, 1981).
These methods are currently employed widely and contributed to immunochemical
investigstion of HB virus as well as to clinical and public health studies.
Also detection of HBs antigen by Orcein staining (Shikata, 1974), HBc antigen
and HBs antigen of formalin fixed liver cells by fluorescent antibody technique
(Yoshizawa, 1977), HBe antigen and immunoglobulin in glomerular capillary walls
of membranous glomerulonephritis (Takahashi, 1978), receptor for polymerized
human and chimpanzee albumins on hepatitis B partcles co-occuring with HBe (Imai,
1979) also contributed to elucidation of phathogenic mechanisms of hepatitis B
virus infection.
In this issue, I will briefly summarize the markers of hepatitis B virus
infection and their detection methods.
(I) Markers of Hepatitis B Virus Infection
According to the taxonomy and nomenclature of viruses, 1982, after the fifth
International Congress of Virology, human hepatitis B virus is now classified
in "hepatitis B virus family" which is distinct from other hitherto known DNA
or RNA virus families. Wood-chuck. Marmota monax, hepatitis virus (WHV), ground
squirrel, Spermophilus beecheyi, hepatitis virus (GSHV) and Pekin duck, Anas
domesticus, type B hepatitis virus (DHBV) are included in this new virus family
The virus consists of nucleocapsid, which is an inner 27 nm, core particle
covered by virus envelop, 7 nm lipoprotein surface layer which has surface (HBs)
antigenic activity. HBs antigen is also-present on spherical particles mesuring
22 nm in diameter with a molecular size about 3 millions. These small particles
are observed most numerously in circulating blood due to over-production of surface
components in HBV infected host and appear to be made up exclusively of HBs antigen
as do tubular or filamentous forms which have the same diameter but may be over
200 nm long.
Two antigenic specificities, a and Re are common to all HBs antigen preparation.
Common antigenicity of a is also found in envelops of WHV and GSHV. In addition,
there are 2 sets of mutually exclusive determinants, d or y and w or r. This
results in 4 principal subtypes of HBsAg: adw, ayw, adr, and ayr.
In HBs antigen, all antigenic determinants and full immunogenicity were
demonstrated in 49,000 dalton poIypeptide (P49), which were cleaved into 22,000
(PI) and 27,000 (PII), which have been known as two principal polypeptides.
Both peptides combined by disulfide bonds show similar amino acid sequencies
and PI1 is glycosylated. They are supposed to be composed of 226 amino acids
coded by gene S region of HBV DNA.
After removal of surface layer (HBs antigen) from Dane particle, core (HBc)
antigen is shown on the naked core particle, which is nucleocapsid of the virus.
The main antigenic component of core particles is polypeptide with molecular
weight 19,000 (P19) composed of approximately 180 amino acids coded by gene C
region of HBV DNA. PI9 has been demonstrated to have both HBc and HBe antigenicity.
After partial digestion of core particles with protease, the HBc antigenicity
was lost and P19 is converted into polypeptide with 15,500 to 16,500 dalton
(P15.5) which has only HBe antigenicity. P15.5 is also the component of circulating
HBe antigen in the serum which is present in free or IgG-bound form and reflects
high infectivity of HBV in the blood. The antigenic heterogeneity of HBe antigen
(el, e2 and e ) remain unclear but e has two antigenic sites ( a and b sub5 3 specificities) which are demonstrated by monoclonal antibodies.
DNA polymerase activity and endogenous DNA template, as well as protein
kinase activity are shown in the inner core particle. DNA template, HB virus
genome, is circular double stranded DNA with a molecular weight of approximately 6 1.6 x 10 , composed of - L chain, approximately 3200 bases length with a nick
at a position of 1818 and + S chain with a length of 50-85 % of L chain, of
which 5' terminal is situated at a fixed nucleotide of - L chain. Some 250-300
nucleotides of 5' terminals of both chians are cohesive and maintained the circular
form of HBV DNA. Therfore 50 to 15 % of viral circular DNA is single strand
and DNA polymerase activity is to repair the single strand portion starting from
3' terminal of + S chain into double ~tranded DNA. DNA polymerase is coded by
gene P region of HBV DNA and measurement of this enzyme activity give5 a good
quantitative marker of HBV infectivity as well as HBV DNA itself. Infectivity
of the virus is not completely lost by heating at 60°C for 10 hours but lost
by heating at 9 8 " ~ , 2 minutes and by treatment with 1 :2,000 dilution of formalin
- 2 -
at 37°C for 96 hours in diluted plasma. For disinfection, autoclaving, boiling
for 10 minutes, treatment with 0.1 % Na-hypochlorite (e-g. 1:50 clorax), 2 %
glutar aldehyde or ethylen-oxide gas are practical and washing out the contaminated
materials throuehlv with running tap water to dilute out the virus infectivity
is recommended.
HBV DNA was cloned in E. coli and DNA sequence was determined in 1979.
HBc antigen was produced by E. coli and HBs antigen was produced by yeast
employing recombinant DNA techniques. Production of test reagents and vaccine
with these methods became possible. In this way, both immunochemical investigation
and molecular characterization of the viral DNA have elucidate the nature of the
HBV infection standing upon these backgrounds.
(11) Laboratory Tests for the Detection of HBV Markers
Routine laboratory tests of HBV markers are classified into three steps.
First screening: HBs antigen This is for detection of possibility of the presence
of HBV.
Second step: (a) Subtype of HBsAg for pursuing the route of infection, (b) HBeAg,
anti HBe, DNA polymerase activity, HBV DNA, for estimation of the infectivity
of the HBs antigen positive blood or assume the prognosis of clinical c.ourse.
(c) Anti HBcAg. To differentiate current infection and remote infection. IgM anti
HBc is an early antibody and is probably associated with viral replication. The
ratio of IgM to IgG anti HBc will give better marker for distinguishing both
types of infection.
Third step: Anti HBs. This is useful for the purpose of epidemiological survey
of HB virus spread and contributes as a marker of immunological host resistance
against HBV infection but not for clinical diagnostic value for viral hepatitis
especially with IgG type anti HBs.
In addition to the laboratory test erllploying~er~l~gi~al measurement of
circulating HBV markers, biopsy 0rautoDsy:spe~imen can be used for detection of
HBs antigen or HBc antigen in liver tissues. Especially formalin fixed specimen
after storage for several decades can be used for detection of both HBs antigen
and HBc antigen in liver tissues.
Based on the laboratory tests, HBV infection can be classified into (i)
transient HBV infection and (ii) persistent HBV infection.
(i) Transient HBV infection (acute type)
Upon primary exposure of the virus to immunologically matured host,
acute type of HBV infection occurs. In this case, transient antigenemia of both
HBsAg and HBeAg is followed by clinical manifestation of hepatitis with rather
long incubation period. Then antibody responses against HBcAg, HBeAg and finally
anti-HBsAg are induced resulting in elimination of the virus and healing of
hepatitis. IgM anti HBc appeared earlier and IgG anti HBc later. In current stage
shortly after primary exposure, the ratio of IgMjIgG anti HBc is much higher than
promate stage.
Most cases of acute type hepatitis B, post transfusion hepatitis, accidental
hepatitis in medical institutions and infection through drug abuser of venereal
routes are this trpe of infection. Except few cases of fulminant hepatitis, the
prognosis of this type of infection is better generally and development of chronic
hepatitis, liver cirrhosis or hepatoma are extremely rare. Of course, many cases
of subclinical infection exist and they are recognised by demonstration of anti
HBs antibody.
(ii) Persistent HBV infection (chronic type)
The second, persistent HBV infection is characterised by persistent
HBs antigenemia, persistent IgG anti HBc with high titre over 1000 by IAHA unit
and very rare anti HBs response. Clinical symptoms of hepatitis are often observed
as exacerbation of persistent HBV infection with positkve HBeAg. In some of such
cases, differential diagnosis from the first type, i.e. acute hepatitis due to
HBV transient infection, is 1 difficult clinically but through measurement of these HBV markers as described above, especially the ratio of IgM/IgG anti HBc
titre, two types can be differentiated.
HBV infection of this type occurs by primary exposure of the virus to
immunologically inmature or impaired host to HBs antigen and specifec immunological
unresponsiveness to HBs antigen occurs. Therfore, coexistence of HBV without
immune elimination from the host is induced in the host exposed to HBV. This
type of infection occurs in the newborn babies born to the HBe antigen positive
HBV carrier mothers mostly at the time of birth and small portion in utero.
Close contact with HBV carriers in childhood is also considered as main routes
of infection. Immunosuppresive effect due to virus infection such as measles,
rubella and other diseases might cause immunological unresponsiveness to the
exposed HBV resulting in induction of HBV persistent infection.
1) l.ests for Hepatitis B Surface Antigen and its Antibody
HBsAg and anti-HBs, the first antigen-antibody system to be associated with
hepatitis, were originally detected by immunodiffusion, a simple test which allows
for high specificity, but relatively low sensitivity. Many other methods are now
available for their detection. Somewhat more sensitive techniques include counter-
immunoelectrophresis, rheophoresis, comlemen-fixation, haemagglutination, and
immunofluorescence tests. Greater sensitivity for detection of HBsAg has been
achieved by immune adherence haemagglutination, reversed passive latex agglutination,
reversed passive haemagglutination, and immunoelectronmicroscopy. RPHA has been
recommended by WHO scientific group as a most practical method with a greater
sensitivity and specificity for detection of HBs antigen. For detection of HBs
antibody, PHA technique has been considered as most practical laboratory test for
anti HBs antibody detection. The most sensitive methods for detecting HBsAg or
anti-HBs are radioimmunoassays(RIA), including solid-phase and radioimmunoprecipi-
tation techniques; the latter being a double antibody RIA. More recently, enzyme
immunoassays have been shown to possess a sensitivity similar to that of radio-
immunoassays. Positive test results obtained with the highly sensitive technique
should be confirmed as specific by appropriate means. A variety of HBsAg subtypes
have been identified first by immunodiffusion, followed by hemagglutination
inhibition test with greater sensitivity. Recently solid phase RIA employing
monoclonal antibody was developed.
Immune complex of HBs antigen, which may play a role in pathogegis;-rnerbanis~;,;~
of active viral hepatitis can be measured by immune adherence of immune complex
with complement to C3b receptors of human erythrocytes followed by coupling with
radio-labelled anti HBs antibody.
Co-existence of antibody against different subdeterminant of HBs antigen
was observed in some cases of carriers of HBs antigen, such as anti-w antibody in
HBs/adr carrier. This is not immune con~plex of HBs antigen and antibody.
2) Tests for Hepatitis B Core Antigen and its Antibody
Uncoated hepatitis B core antigen (HRcAg) has not been detected in the circulation
but it is present in the nuclei of infected hepatocytes where it has been
identified by various techniques.
Anti HBc antibody can be measured by hemagglutination inhibition test,
immune adherence hemagglutination, RIA and enzyme immunoassay. Hemagglutination
inhibition and routine radioimmunoassay detect both anti HBc IgM and anti HBc IgG,
while immune adherence hemagglutination detects anti HBc IgG only. Radioimmunoassay
using beads coated by human IgM antibodies is suitable for detecting IgM anti
HBc . Anti-HBc IgM is present during the acute and early convalescent stages of
uncomplicated acute type of HBV infection. In cases of chronic infection,..;rnti-
HBc IgM levels may indicate the degree of virus activity. In contrast, low titres
of only anti-HBc IgG in the absence of other serological markers of HBV may
indicate a previous HBV infection; these possibilities should be considered in
epidemiological investigations'. High titre of only anti-HBc IgG, most preferably
measured by IAHA may indicate a persistent infection of HBV.
3) Tests for Hepatitis Be Antigen and its Antibody
The presence of HBeAg in the serum appears to correlate with the replication
of hepatitis B virus in the host and may be of some prognostic significance.
There is a highly significant correlation between HBeAg, 'DNA polymerase, circulating
HBV and infectivity. Anti-HBe is associated with reduced or relatively low
infectivity; virus production can occur in the presence of anti-HBe, although
at a diminished level.
Several serological methods are now available for the detection of HBeAg
and anti-HBe. In order of increasing sensitivity these include immunodiffusion,
rehophoresis, immunoelectrosyneresis, passive haemagglutination, enzyme immuno-
assay and RIA. HBe antigen exists in the serum in dual forms. One form is smaller
than IgG, soluble in 1.33 M ammouniwn sulfate, electrophoretic mobility in d-globulin
region and the other is larger than IgG, insoluble in 1.33 M ammonium sulfate
and electrophoretic mobility in fl- to $-globulin regions. The first is free
"small" HBe antigen and the latel' is IgG-bound "large" HBe. Bcth can be differentiated
using these characterstics with sensitive method for detection of HBe antigenicity
such as passive hemagglutination or RIA.
There is a shift from free HBe antigen through IgG bound HBe antigen to anti-
HBe stages in acute type of HBV infection or chronic type of HBV infection as well
as in expermental infection in chimpanzee. IgG bound HBe was also found in the
patients with membranous glomerulonephritis. Immune complex composed of HBe antigen
and antibody may play a role in various allergic syndrome with HBV infection.
Conversion of HBe antigen to anti HBe kcas not only observed in natural course
of infection but also in the course of anti-viral treatment of chronic HBV infection
and might give a proper monitoring system for evaluation of anti-viral treatment.
4) Tests for Hepatitis B Virus DNA Polymerase
Special assays detect this enzyme activity which can complement other HRV
serological tests or it may correlate with increased infectivity. A close correlation
has been demonstrated between the presence of specific DNA polymerase and infectious
hepatitis B virus. The detection of specific DNA polymerase is therefore a useful
marker of HBV replication. This enzyme activity is often found early in the course
of infection when large numbers of virus particles are present. It generally
correlates with the presence of HBeAg and it persists in some chronically infected
individuals with continuing viral replication. Frequent fluctuations in the levels
of DNA polymerase are observed in serial samples from chronically infected patients
and such variations should be taken into account while monitoring the effect of
treatments to alter the chronic carrier state.
5) Tests for HBV DNA
After development of HBV-DNA cloning in E. coli, probe of 3 2 ~ labelled HBV
DNA can be utilized to detect HBV DNA in fresh liver tissue frozen in liquid
nitrogen. Liver DNA was extracted and digested with a restriction endonuclease.
DNA fragments were fractionated by agar-gel electrophoresis, denatured and
transferred to a nitrocellulose filter. The denatured DNA fragments immobilized
on the filter were hybridized with denatured cloned 3 2 ~ labeled HBV DNA. Hybridi-
zation which indicates HBV DNA in liver tissue is revealed by the presence of
bands on autoradiogram. The determination of the state of HBV DNA in the liver
cells, whether integrated into host cell DNA or episomal state can be done
employing proper restriction enzymes. Measurements of HBV DNA in the serum corres-
ponds well with the level of DNA polymerase activity and 1 picogram of HBV DNA 5 6 (5 x 10- - 10 HBV particles) can be measured. Although important for molecular
and pathogenesis studies with great sensitivity, these techniques are not used
for routine diagnosis of HBV infection at present.