Download Atypical serological profiles in hepatitis B virus infection Robério A

Atypical serological profiles in hepatitis B
virus infection
Robério A. A. Pondé
European Journal of Clinical
Microbiology & Infectious Diseases
ISSN 0934-9723
Eur J Clin Microbiol Infect Dis
DOI 10.1007/s10096-012-1781-9
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Author's personal copy
Eur J Clin Microbiol Infect Dis
DOI 10.1007/s10096-012-1781-9
REVIEW
Atypical serological profiles in hepatitis B virus infection
Robério A. A. Pondé
Received: 27 September 2012 / Accepted: 12 November 2012
# Springer-Verlag Berlin Heidelberg 2012
Abstract During hepatitis B virus (HBV) infection, at least
four antigen–antibody systems are observed: HBsAg and
anti-HBs; preS antigen and anti-preS antibody; HBcAg
and anti-HBc; and HBeAg and anti-HBe. Through the examination of these antigen–antibody systems, hepatitis B
infection is diagnosed and the course of the disorder may
be observed. Although the serologic findings that allow both
the diagnosis of HBV infection as well as assessing of its
clinical course are already well established, the dynamics of
viral proteins expression and of the antibodies production
may vary during the infection natural course. This causes the
HBV infection to be occasionally associated with the presence
of uncommon serological profiles, which could lead to doubts
in the interpretation of results or suspicion of a serological
result being incorrect. This paper is dedicated to the discussion
of some of these profiles and their significance.
Introduction
The natural course of hepatitis B virus (HBV) infection is
determined by the inter-relationship between viral replication via HBV protein production and the host’s immune
R. A. A. Pondé
Laboratório de Virologia Humana, Instituto de Patologia
Tropical e Saúde Pública, Universidade Federal de Goiás,
Goiânia-Goiás, Brazil
R. A. A. Pondé
Gerência de Hepatites Virais,
SUVISA - Superintendência de Vigilância em Saúde - Secretaria
Estadual de Saúde, Goiânia-Go, Brazil
R. A. A. Pondé (*)
Sorologia e Biologia Molecular, Central Goiana de Sorologia,
Rua 7A Edifício RIOL,
N° 158, 1° andar, sala 101, Setor aeroporto,
Goiânia-Goiás 74075-030, Brazil
e-mail: [email protected]
response, and, therefore, clinical practice diagnosis of
HBV infection is established by the serological detection
of HBV protein products (antigens) as well as hostproduced antibodies. During HBV infection, four structural
antigen–antibody systems are observed: hepatitis B surface
antigen (HBsAg) and its antibody (anti-HBs); the preS antigens associated with HBsAg particles and their antibodies;
the particulate nucleocapsid antigen (HBcAg) and anti-HBc;
and an antigen structurally related to HBcAg, namely, hepatitis B e antigen (HBeAg) and its antibody (anti-HBe).
Through the examination of these antigen–antibody systems, hepatitis B infection is diagnosed and the course of
the disorder may be observed
Classically, 8–9 weeks following the infection, in the
incubation period or 3–5 weeks before biochemical evidence of liver dysfunction and the appearance of clinical
symptoms, it is possible to detect HBsAg in serum. HBsAg
detection is used for the diagnosis of acute and chronic HBV
and indicates potential infectiousness. In patients who subsequently recover from HBV infection, HBsAg usually
becomes undetectable after 4–6 months [1]. If HBsAg persists for more than 6 months, spontaneous clearance is very
improbable and the infected individual is considered to be a
chronic HBV carrier [2].
However, during the acute phase of infection, following
HBsAg detection, other viral markers can be easily detectable, including DNA polymerase and HBeAg. HBeAg
appears shortly after the appearance of HBsAg and disappears within several weeks as acute hepatitis resolves. Its
presence in the serum correlates with the presence of viral
replication in the liver and HBsAg detection, while its
disappearance, associated with the anti-HBe detection, is
seen as a sign of the absence of viral replication and spontaneous resolution of acute infection.
Anti-HBc IgM antibodies are detectable at the outset of
clinical disease, and, as the infection evolves, IgM anti-HBc
levels gradually decline, often becoming undetectable
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within 6 months and IgG class predominates, remaining for
a long period of time (sometimes life-long) at detectable
levels [2]. The presence of IgM anti-HBc usually indicates
recent or continuous HBV replication, while the presence of
IgG anti-HBc correlates with past infection [3].
As recovery and convalescence signal, antibodies to other viral proteins appear in the blood. Anti-HBe antibodies
appear after HBeAg clearance, and may persist for many
years after the resolution of acute HBV infection. In some
cases, the phenomenon of “immunological window” of the
HBe-anti-HBe system can be observed, with anti-HBe antibody detection about 2 months after the disappearance of its
antigen precursor.
The antibody response to hepatitis B surface antigen (antiHBs) becomes detectable in convalescence, from 6 weeks to
6 months after HBsAg clearance, although it is produced early
in the course of an acute infection. Its detection indicates
infection recovery. The antibodies to HBsAg are able to
neutralize HBV infectivity and, therefore, clear circulating
HBsAg and infectious HBV particles from peripheral blood.
These neutralizing antibodies are especially important in the
prevention of viral infection, since they could prevent viral
attachment and entry into the cells by absorption of the viral
particles [3]. Its presence is considered to be an indicator of
immunity to HBV infection, although it could become undetectable in patients who have recovered fully from infection
(for a review, see [4]). Table 1 shows the dynamics of the
expression of HBV serological markers, the corresponding
antibodies, and the interpretation of the profile, in accordance
with the evolution in an typical serological profile, during the
natural course of infection.
Currently, the most accurate tool available for the diagnosis of HBV infection is DNA detection, usually obtained
by genomic amplification assays. However, anti-HBVspecific tests are used routinely: (i) to assess disease activity
in persistent infection; (ii) in monitoring therapeutic regimens with antiviral agents, and, especially, (iii) in an attempt
to diagnose the presence of infection and its development,
according to the dynamics described above. Although the
serologic findings that allow both the diagnosis of HBV
infection as well as assessing its clinical course are already
well established, the dynamics of viral proteins expression
and of the antibodies production may vary during the infection natural course, due to factors related to the agent (infection by a new strain or viral serotype—antigenic variant)
and/or to the host (immune tolerance, cellular immune response, and immunosuppression) [5–7]. This causes the
HBV infection to be occasionally associated with the presence of uncommon serological profiles, which can lead to
doubts in the interpretation of results or suspicion of an error
in the serological diagnosis. The purpose of this paper is to
discuss the atypical profiles most frequently found in HBV
serology.
Profile 1
Isolated positivity for HBsAg and detectable DNA
HBsAg [+]; HBeAg [+/−]; anti-HBc [−]; anti-HBe [−];
anti-HBs [−]; DNA/HBV [+]
The isolated positivity for HBsAg comprises a finding often
observed in HBV serology whose ample meaning was discussed recently [8]. Accordingly, this serological profile, associated with DNA detection in the bloodstream, suggests,
initially, acute infection, which is a consequence of recent
exposure to HBV, and this hypothesis, in most cases, can be
confirmed after the subsequent expression of other markers
detected by laboratory testing. However, the persistent isolated positivity for HBsAg and detectable DNA, in the absence
of other markers, comprises an atypical serological profile,
whose presence may indicate immune tolerance to HBV core
antigen (an abnormality of the host’s immune system) or
Table 1 Schematic representation of hepatitis B virus (HBV) serological markers, the corresponding antibodies, and the interpretation of the
profile, considering the typical serological profiles in HBV infection
HBsAg
HBeAg
HBcAg IgM
HBcAg IgG
Anti-HBe
Anti-HBs
Interpretation
POS
POS
POS
POS
NEG
NEG
NEG
NEG
NEG
NEG
NEG
POS
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
POS
NEG
NEG
POS
NEG
NEG
NEG
NEG
NEG
NEG
POS
POS
POS
POS
POS
POS
POS
NEG
NEG
NEG
NEG
NEG
NEG/POS
NEG
NEG
POS
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
POS
POS
POS
NEG
Incubation period
Acute infection
HBV infection (acute or chronic HBV infection)
Chronic HBV infection or end of recent infection
Recent HBV infection. Beginning of the convalescence period
Past HBV infection (immunological window)
Immune, recent past infection
Immune, past infection (old infection)
Immune, contact HBsAg, vaccine response
Susceptible individual. Never had contact with HBV
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suggest infection by an HBV mutant, factors that can compromise anti-HBc antibodies production.
The immune tolerance to HBcAg is known by the incapacity of the individual to produce anti-HBc or to produce it in
undetectable levels. The immune tolerance can be mediated
by a variety of mechanisms. For example, T-cell anergy is a
tolerance mechanism in which the T cell is functionally inactivated following an initial antigen encounter, but remains
alive in a hypoactivated state [9]. T-cell anergy has been
proposed as a major mechanism for the maintenance of selftolerance and regulation of the cellular immune response [10].
In addition, a small number of specific peripheral T lymphocytes, an inefficient antigen presentation, or lymphokine production by the antigen-presenting cell are, among others,
possibilities associated with anti-HBc production [11]. Therefore, a selective immune system defect could lead the immune
tolerance to the HBc and, consequently, the lack of production
of corresponding antibody, justifying, therefore, the existence
of this serological profile [12, 13].
The phenomenon of the immune tolerance to HBc antigen has also been associated with the vertical transmission
of infection, and, consequently, the absence of an antibody
response to HBc in infants born to HBe-positive carrier
mothers [14]. It has been postulated that HBeAg may have
an immunoregulatory function in promoting viral persistence [15]. The HBcAg and HBeAg are distinctly recognized by antibodies but, due to their extensive amino acid
homology, are highly cross-reactive at the T-cell level [15].
Thus, HBe antigen, because of its small size, may traverse
the placenta and elicit HBe/HBcAg-specific T-helper cell
tolerance in utero [16]. Consequently, it may lead to fetal
immunotolerance not only to HBeAg but also to HBcAg,
and still inhibit anti-HBc antibody production when it is
present in the serum.
As mentioned earlier, it is possible that agent-related
factors can also justify the anti-HBc non-production by the
host and contribute to the expression of this serological
profile. Among them include infection by HBV which produces a non-responsive HBc [17, 18], therefore, unable to
induce an immune response, or by HBV mutant with nucleotide sequence deletions in the core gene. These deletions
may involve important epitopes to antigen recognition by
immune response cells, and, therefore, compromise the HBc
antigenicity, becoming a non-functional protein [19, 20].
The presence or absence of HBeAg in this profile may be
inconsistent or even vary from case to case, since its detection (or not) will be associated with the mechanism that
justifies the profile in question. For example, in the case of
immune tolerance specific to c antigen or due to vertical
transmission [12, 13], the HBeAg detection in the profile it
seems likely, since, in these cases, there is no compromising
of the viral antigens expression (the preC/C regions remain
unchanged, with normal synthesis of viral antigen). On the
other hand, following the occurrence of core gene deletions,
depending on the region involved, the HBeAg expression
may become compromised, since both antigens are encoded
by the same gene.
Additionally, it is of great interest, and deserves highlight, the observation of this serological profile in the chronic infection context. Serologically, the chronic HBV
infection is characterized by persistence of HBsAg in the
bloodstream for more than 6 months, associated with the
presence of anti-HBc antibodies and detectable DNA. During the natural course of chronic infection, the spontaneous
seroclearance of HBsAg and even of DNA can occur after
antiviral therapy, but anti-HBc antibodies remain detectable
continually in peripheral blood [21]. However, in some
circumstances, the presence of anti-HBc antibodies may
not be observed in this clinical context, but only isolated
HBsAg positivity and DNA detectable in the serological
profile, comprising an unusual finding in medical practice.
In this case, the serological pattern is justified by immune
complexes formation of anti-HBc antibodies with an excess of
HBc antigen in the bloodstream, with anti-HBc becoming
undetectable [22]. These immune complexes cannot be
detected by available commercial tests, which are not able to
detect antibodies in the presence of excess antigen in the serum
[23]. Anti-HBc complexes could be detected only after immune
complex dissociative treatment. Some methods are based on
polyethylene glycol precipitation followed by ion chaotropic
treatment [24] or based on sucrose gradient fractionation [25].
HBcAg is not a secreted protein and exists primarily in
liver and in serum within HBV particles, not being directly
accessible in the blood to the immune system. In exceptional
cases, HBc epitopes may, however, be exposed to virions
present in the serum [26] and also during liver necrosis,
when nucleocapsids may be secreted from infected hepatocytes with a high level of HBV particles, explaining the
presence of anti-HBc complexes in the bloodstream. This
phenomenon has been observed in chronic HBV carriers
during the phase of active replication in a state of immunosuppression, as seen in the context of transplantation (bone
marrow, kidney, heart), during chemotherapeutical treatment
for neoplasias [27], or an uncontrolled human immunodeficiency virus (HIV) infection, conditions which could also
lead to an immune system defect. HBcAg is the most
immunogenic HBV component and induces the production
of high titers of anti-HBc antibodies in immunocompetent
individuals who have been exposed to HBV [28]. However,
the immunosuppression conditions can potentially induce
decreased antibody production [3]. HBcAg-reactive material is released from HBV particles present at a high level in
the serum and from hepatocytes undergoing lysis. Consequently, this could complex to low levels of anti-HBc produced in these patients and, therefore, lead to the nondetection of antibodies (Fig. 1).
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Phase of active
Chronic HBV carrier
Hepatocyte lysis
replication
HBsAg [+] / HBV free
Anti-HBc [+] [free] / HBeAg [+/-]
Anti-HBc antibodies
Immune-complex [IC]
Immunosuppression
[HBcAg-anti-HBc]
[anti-HBc in low levels]
HBcAg free
Anti-HBc [+] [not complexed]
Anti-HBc [ - ] and HBsAg [+]
[in the serum]
HBcAg complexed
HBcAg [free] / Anti-HBc-HBcAg [IC]
HBV
HBc Antigen
Anti-HBc-HBcAg [IC]
HBsAg
Fig. 1 Schematic representation of isolated positivity for HBsAg in
the chronic hepatitis B virus (HBV) infection context, due to the
formation of immune complexes HBcAg and anti-HBc. In chronic
HBV carriers, during the phase of active replication, hepatocyte lysis
occurs, as well as the release of HBcAg in the bloodstream, and
subsequent complexation with anti-HBc free in excess. In the immunosuppression conditions (and low levels of anti-HBc antibodies), after
hepatocytes lysis, the HBcAg released (in excess) complex with antiHBc antibodies, leading to the non-detection of antibodies and isolated
positivity for HBsAg in the serological profile
Profile 2
molecular biology techniques [33]. However, little is known
about the molecular mechanism of viral persistence in these
cases and the non-detection of HBsAg in this serological/
molecular context.
The molecular basis of the occult infection seems to be
strictly related to the peculiar life cycle of HBV. In particular, a fundamental step is the conversion of the relaxed
circular genome into a covalently closed circular DNA
(cccDNA). This comprises a long HBV replicative intermediate that persists in the cell nuclei (hepatocyte) as a stable
episome and that serve as the template for mRNAs and
RNA pregenomic transcription [34]. During the viral replication cycle, viral transcript is translated in capside, together
with polymerase proteins. After subsequent encapsidation,
RNA pregenomic is retranscripted into new partial doublestrand viral genomes. The new nuclear capside containing
DNA may be secreted as a complete virion after the attachment of coating protein or, then, may redirect the viral
genome to the hepatocyte nucleus to pool the maintenance
of cccDNA and to retain permanently infected cells [31, 35,
36]. Thus, considering the stability and long-term persistence of viral cccDNA molecules in the liver tissue, it seems
plausible to affirm that, (i) HBV infection, once it has
occurred, may possibly continue for life, and (ii) resolution
of disease does not imply complete eradication of infection,
but, more likely, reflects the capacity of the host’s immune
response to restrain HBV and to keep persistent control of its
replication [33, 35].
Accordingly, histopathological analysis of the liver by
immunohistochemistry in individuals with occult infection
has revealed the expression of HBsAg or HBcAg in the liver
tissue comparable to those of chronic HBV carriers, despite
the signals being lower [37, 38]. Additionally, the presence
of extrachromosomal HBV/DNA and pregenomic RNA in
the liver and peripheral blood mononuclear cells by molecular techniques [polymerase chain reaction (PCR) and in
Positivity for anti-HBc antibodies, with or without detection
of anti-HBs antibodies and detectable DNA
HBsAg [−]; anti-HBc [+]; anti-HBs [+/−]; DNA/HBV [+]
Against the classical information that the anti-HBc detection
in association with anti-HBs antibodies in the bloodstream
is indicative of viral clearance and immunity, this serologic/
molecular profile is indicative of persistent infection and has
been associated with the presence of occult HBV infection.
The anti-HBc antibodies detection is justified by the high
immunogenicity of the core antigen [29] and includes a
classic finding in cases of exposure to HBV. On the other
hand, the anti-HBs antibodies (when present), in the context
of this serological/molecular profile, can be of heterologous
nature (i.e., directed to another HBsAg antigenic determinant) or be directed to HBsAg (mutant or not, but not
detectable) of the first viruses infecting, which does not
recognize, however, the persistent HBV, not having, therefore, any protective or neutralizing activity of the infection
developing [30]. The generation mechanism of anti-HBs
antibodies and its significance will be discussed in detail
in “Profile 4”. This discusses the non-detection of HBsAg in
the context of occult infection, justifying the serological/
molecular profile in question.
Occult infection can be defined as the long-lasting persistence of viral genomes in the liver tissue (and, in some
cases, also in the serum) of individuals negative for the
HBV surface antigen [31]. In many instances, occult hepatitis B is associated with hepatitis B core antibody (antiHBc) and/or anti-HBs [32]. In this context, the viral genome
persistence under conditions not yet elucidated is characterized by a strong suppression of viral replication and expression of its genes, identified only by highly sensitive
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situ hybridization] has been demonstrated in some patients,
suggesting the maintenance of transcriptional activity, and,
therefore, HBV activity [39]. Finally, in the chronic infection context, the persistence of replicative forms of the viral
DNA in the hepatocytes nucleus, after HBsAg disappearance (spontaneously or due to a successful treatment), has
also been detected by in situ hybridization, despite the
inhibition of DNA synthesis by antiviral therapy [40]. These
data illustrate that, (i) it is possible that some patients, even
after the loss of serological markers of active infection, may
still demonstrate a low degree of infection with liver disease
development, despite the antibodies presence, and (ii) the
persistent infection can occur without the production of viral
particles. The frequency with which this occurs is unknown,
although there are reports which have indicated that 3 out of
every 16 monitored patients after acute infection remain
viremic for a period of several years [41, 42].
Even considering the low levels of replication and intrahepatic HBV transcription maintained during the natural
history of occult infection in these individuals, a question
remains open. What reasons would justify the nondetection/expression of HBsAg in this situation? Two possibilities have been discussed: the antigen may be absent
from the peripheral blood or may be present but not detectable. In the first situation, the absence of antigenemia could
be due to mutations characterized by deletions in the preS
region [43] or in promoter sequences of the HBV genome
which would block HBsAg secretion, causing its accumulation inside the cell [38, 44]. In the second situation, the
HBsAg may be present but, however, escaping, the recognition by enzyme immunoassays currently standardized,
depending on the sensitivity of detection [45]. There is,
however, an additional explanation for the non-detection
of HBsAg: mutations in regions that code for protein S
[46]. Specifically, mutations in the major hydrophilic region
of HBsAg, the main target for antibodies used in diagnostic
tests. These mutations could lead not only to an escape from
recognition by the tests routinely used, but may also reduce
the replicative capacity of the virus, reducing the levels of
HBsAg, which could compromise its detection in the bloodstream. Bréchot et al. [47], however, have provided strong
evidence that most cases of occult HBV is more related to
low levels of HBV wild-type (low viral load) than the
presence of HBV mutants that do not express surface proteins or that produce with conformational structure changes.
Concerning the absence of anti-HBs antibodies in the
serological profile, the failure to detect these antibodies in
many patients in this context does not necessarily indicate
the absence of humoral immune response, and may be
justified by the formation of immune complex HBsAg
anti-HBs. As a practical proof of this fact, it has been shown
that anti-HBs can be detected in all the patients that evolve
into chronic infection, when measured by an assay able to
detect an antibody in the form of immune complexes [23].
Another explanation for failure in detecting anti-HBs could
be the low sensitivity of commercial assays currently standardized, which are not adjusted to detect antibodies produced in low levels, since an increase in its sensitivity would
also increase the risk of false-positive results [48, 49]. This
observation suggests that the marker detection depends on
the sensitivity of the test adopted and of the detection limit,
which is arbitrarily determined by the manufacturer [49, 50].
Finally, the occurrence of residues substitutions in
HBsAg, involving specific epitopes, could be able to prevent the protein recognition by T-cells CD4+ or minimize
the ability of its stimulation, compromising, consequently,
the anti-HBs production [51, 52] and its detection in the
serological profile.
In conclusion, in this clinical setting, the detection or not
of anti-HBs antibodies in the serologic profile HBsAg [−];
anti-HBc [+]; anti-HBs [+/−]; DNA/HBV [+], does not
seem to have great significance and implications, since these
antibodies, in detectable levels, do not fulfill its function of
neutralizing, whereas its presence below detection levels,
despite characterizing the presence of immune response, is
insufficient to prevent viral replication and establish infection control.
Profile 3
HBeAg positivity/negativity for HBsAg
HBsAg [−]; HBeAg [+]; anti-HBc [+]; DNA/HBV [+]
HBeAg is a secretory protein that is processed from the
precore protein of HBV. It is generally considered to be a
marker of HBV replication and infectivity, although it is not
required for infectivity or viral replication [53]. As a nonstructural component of HBV, HBeAg is abundantly secreted into the bloodstream during the acute phase of infection,
appearing shortly after the appearance of HBsAg. Classically, the HBeAg presence in the bloodstream correlates with
high levels of viral replication in hepatic tissue and HBsAg
detection in serum. Therefore, HBeAg positivity/HBsAg
negativity in the serological profile comprises an atypical
pattern that deserves to be addressed.
The ‘a’ determinant of HBV consists of a defined serological region, located between amino acid residues 124 and
147 of the HBsAg, which induces a protective immune
response common to all HBV subtypes [54]. Variations in
its primary structure have been demonstrated to markedly
alter the antigenic conformation and antigenicity of HBsAg.
For example, the loss of the proline at position 120 significantly minimizes the binding of antibodies mapped to that
region, as does the insertions between residues 122 and 124
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[55]. A similar effect is the occurrence of substitutions at
positions 122 and 123, which demonstrate that this region is
a critical site in the HBsAg antigenicity [56]. In vitro studies
have shown that the HBsAg antigenicity is also lost when
cysteine residues at positions 124, 137, 139, 147, or 149 are
replaced by serine, suggesting that this amino acid is of
fundamental importance in maintaining the conformational
structure of the molecule [57]. Thus, the maintenance of the
tertiary molecular structure of HBsAg, especially of its
immunodominant region, is of crucial importance to recognition by antibodies, since antibodies directed to this region
protect against HBV infection, and HBV variants that are
not recognized by them can escape its protective action [58].
On the other hand, the commercial tests usually detect
this protein using a capture monoclonal antibody and a
detection monoclonal antibody (or polyclonal) anti-HBs
wild-type, such as those produced after vaccination [45,
46]. The antibodies bound to the antigen captured tend to
be specific for epitopes present in the determinant ‘a’ of
HBsAg and oriented to recognize its cyclic peptide sequence [45, 54]. Since the immune response to HBsAg is
primarily directed against conformational epitopes of the ‘a’
determinant, and that its antigenicity has been employed for
HBsAg detection in the serum [54, 59], changes in its
tertiary structure could not only prevent the binding of
neutralizing antibodies targeted to the wild-type virus, but,
also, the antigenic detection by the assay used for diagnosis
[58, 60, 61] (depending on the epitopes recognized by the
test configuration [62]), leading to false-negative results,
justifying, therefore, the serological profile HBeAg [+]/
HBsAg [−] and detectable DNA.
Recent studies have shown conflicting results between
the tests applied in the detection of HBsAg mutants [63, 64].
This has occurred, presumably, due to differences existing
between the tests applied, related to the analytical sensitivity
(> or <0.1 ng/ml) or associated with the system configuration of capture and detection. With regards to the capture
and detection system, the sensitivity of the tests currently
designed to detect HBV mutants can be variable, being
established between 57 % for testing monoclonal/capture
and 80.6 to 97.4 % for testing polyclonal/capture, which has
been linked to the non-recognition of HBsAg epitopes by
monoclonal anti-HBs antibody used in the test [65] and
shows a significant advantage of polyclonal capture systems. However, it is known that the polyclonal tests do not
guarantee full sensitivity in the HBsAg mutant detection,
which makes it important to incorporate specific antibodies against mutant forms of HBV in commercial tests.
Thus, it should be noted that the test configuration is
not the only factor that predicts the capability to detect
the mutant, but, rather, the ability of epitope recognition
by the reagent used to immobilize or detect the antigen
involved [64].
Profile 4
Simultaneous positivity for HBsAg and anti-HBs
(and detectable DNA)
HBsAg [+]; HBeAg [+/−]; anti-HBc [+]; anti-HBs [+];
DNA/HBV [+]
Concurrent HBsAg and hepatitis B surface antibodies comprise a peculiar serological finding, since it contemplates
components that indicate immunity and, at the same time,
developing infection. However, despite the presence of neutralizing antibodies, this serological profile correlates with
active viral replication and may be observed in acute or
chronic hepatitis B infection, although the mechanisms that
could lead to this serological pattern have not been well
delineated and understood. Its existence has been justified
mainly by the emergence of HBV mutants and the presence
of non-protective anti-HBs antibodies, as was recently discussed [30].
In the acute infection context, the identification of this
profile may be justified by the occurrence of reinfection
with a new HBV strain. This new HBV strain could have
an ‘a’ determinant different from the first HBV infection
(mutant), whose circulating antibodies would not have a
neutralizing action, and, therefore, were not protective. Obviously, in this case, the presence of anti-HBs does not
indicate the removal of HBV and means that HBV carriers
with concurrent anti-HBs still have active virus replication,
suggesting that some of the infecting viruses are surface
antigen mutants.
The emergence of HBV escape mutations has been a
phenomenon associated with the replication mechanism
(see below), and also as a result of a selection process due
to a selective advantage of variants during the course of
HBV infection in a patient (for a review, see [6, 7, 66]).
Additionally, the primary prevention of HBV infection, such
as vaccination in infants born to HBsAg-positive mothers
and immunoprophylaxis in orthotopic liver transplantation
recipients, due to exerting strong selective immune pressure
on HBV, in some cases, can also induce escape mutations
within the ‘a’ determinant region, affecting several amino
acid positions, as previously mentioned [67–70]. HBV
mutants can be extremely stable and maintain their ability
to replicate at high titer in the presence of anti-HBs [67].
They can also be transmitted horizontally to other
humans, and, therefore, justify cases of HBV reinfection/
superinfection [71]. HBV reinfection comprises a common
phenomenon in the liver transplantation context, reaching
rates of more than 90 % if there is no treatment [72]. With
the use of LMV and HBIG for prophylaxis, these rates
can be reduced to almost 3.0 %, as demonstrated by Wang
et al. [73].
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Cases of HBV infection in previously vaccinated individuals may also occur [74, 75], and justify the co-existence of
HBsAg/anti-HBs serological profile. Obviously, this would
not be a case of HBV reinfection (since the anti-HBs present
in the bloodstream was induced by vaccination), but demonstrates the possibility of replication of HBV (which produces an HBsAg mutant or not, but which is different from
HBsAg vaccine), in the presence of preexisting anti-HBs
antibodies (vaccine-induced), but which is unable to recognize the circulating virus (Fig. 2a).
In the chronic infection context, the observation of this
profile can be justified by the detection of HBV mutants
naturally selected due to existing anti-HBs endogenous response [76–79]. Consequently, some chronic carriers have
HBsAg e anti-HBs; however, the antibody detected is being
directed against HBsAg epitopes not shared by original
HBsAg present in the bloodstream, and, therefore, not protective [80] (Fig. 2b). In this case, the presence of anti-HBs
does not indicate viral elimination and suggests that HBV
carriers with HBsAg and anti-HBs demonstrate active replication. Moreover, the presence of anti-HBs does not exclude the possibility of a new HBV infection or the
reactivation of preexisting HBV infection.
These mutants have been characterized, presenting deletions [80, 81], point mutations [82], and genetic recombination [83] within the preS/S genes, which have been
identified, frequently, in HBV DNA sequences obtained
from the sera of inactive carriers.
Fig. 2 a Schematic
representation of a infection
with a new HBV strain in a
vaccinated individual
(immune), demonstrating nonrecognition and nonneutralizing of the circulating
virus by anti-HBs antibodies
induced by vaccination. b
Schematic representation
showing emergence of HBV
mutants induced by immune
pressure (anti-HBs endogenous). In the chronic infection
context, the HBV/HBsAg exists
in both forms, free and
immunoglobulin-bound,
whereas the anti-HBs antibodies only exist in the form of
immune complex. Due to the
immune pressure (anti-HBs induced), the emergence of HBV
mutants with later HBV seroconversion (to wild-type virus)
and consequent simultaneous
detection of anti-HBs and
HBsAg mutant in the bloodstream occurs
HBV produces three envelope proteins which are all
encoded in the preS/S open reading frame. The major hepatitis B surface (HBsAg) is coded by the S gene. The middle
HBsAg is coded by the preS2 and S genes. The large
HBsAg is coded by the preS1, preS2, and S genes. The
preS sequences exhibit the highest heterogeneity of the
HBV genome [84, 85]. Mutations in the preS region, such
as deletions involving the preS2 translation initiation codon,
could promote the synthesis of an anomalous HBsAg detectable in the bloodstream, but, however, not recognized by
previously anti-HBs antibodies formed against the original
HBsAg [76, 86]. These mutations can arise in the natural
course of HBV infection and have been more frequently
observed in patients with anti-HBs who did not receive
hepatitis B immunoglobulin or vaccine than in those HBV
carriers who do not have anti-HBs. These mutations are
more frequently observed in asymptomatic carriers [86].
Deletion in the preS1 region has also been associated to
the co-occurrence of HBsAg/anti-HBs, being related with
persistent viremia. In a previous study, a deletion of nucleotide 31 of the HBs gene in a patient with anti-HBs seroconversion but who still remained an HBV carrier with a
virus load of 104 DNA molecules/ml of serum was observed. This deletion led to frame-shift and introduced a
stop codon after the 21 amino of HBs. The resulting HBsAg
lacking the major epitopes recognized by specific antibodies
could favor ongoing viral replication, despite the presence
of anti-HBs [81].
a
Vaccinee individual (immune)
Infection [New HBV strain]
Acute infection/viremia
Anti-HBs antibodies [+]
HBsAg [+] / HBV free
HBsAg [+] [New HBV strain]
[vaccine-induced]
[unrecognized]
Anti-HBs [+]
Anti-HBs antibodies
New HBV strain
New HBsAg
b
Immune pressure
Chronic Infection
Mutation
Seroconversion
HBV/HBsAg [+] free
HBV/HBsAg [+] mutant
Anti-HBs [+] free [detectable]
Anti-HBs [+] not detectable
(free)
HBsAg [+] mutant [free]
HBV
HBsAg
HBV/HBsAg immunoglobulin-bound
HBV Mutant
HBsAg Mutant
Anti-HBs free
[anti-wild type]
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Eur J Clin Microbiol Infect Dis
Besides deletions, and as aforementioned, variations in
the primary structure of the ‘a’ determinant have also contributed to justifying the presence of this profile, since these
variations may markedly alter the antigenic conformation
and antigenicity of HBsAg. Therefore, the binding of both
monoclonal and polyclonal anti-HBs antibodies can be affected by a single aa change, and antibody raised to the
wild-type can be bound less well to the HBsAg variant. On
the occurrence of these variations, there could be, then, the
presence of HBsAg mutant and the concomitant presence of
anti-HBs antibodies not bound to the mutated antigen.
It must be highlighted also that an additional cause to
justify the mutants emergence during the natural course of
HBV infection has been linked to the complex mechanism
of viral replication, characterized by the synthesis of viral
DNA through an RNA intermediary, by reverse transcription [87]. As result of this mechanism and due to biochemical processes of the host cell (besides the infidelity of the
viral replication machinery), errors of bases incorporation
into DNA strands may inevitably occur during viral replication. Considering the large number of viral particles generated in an infected person (1011 viral particles per day),
and that the error rate (because of the polymerase reverse
transcription) can be of the order of 1 error per 1010 bases, in
active infection, 1010 base-pairing errors could be generated
per day over the 3,200-bp genome in a given patient [88].
These errors lead to the generation of multiple variant transcripts from a single template, and, consequently, the formation of a quasispecies pool [88]. While these new
sequences are viable, they effectively compete with the wild
type and comprise a material source for the emergence of
mutants in an environment with significant selective immune pressure during the natural course of HBV infection.
In this context, in the proportion that any newly generated
mutation confers selective advantages to the virus, it could
allow the corresponding viral population to express more
than wild-type virus, indirectly contributing to HBV
seroconversion.
Thus, HBsAg/anti-HBs simultaneous detection could occur, suggesting that the acquisition of anti-HBs could be
associated with the emergence of immune escape variants
persisting for several years in association with wild-type
strains. The presence of HBsAg variants would arise naturally
in chronic carriers and should be a viral strategy to evade the
immune system. Therefore, the detectable HBsAg is a mixture
of the wild type and variants, whereas the detectable anti-HBs
are targeted against the wild-type viruses.
The presence of heterologous subtype-specific antibodies
could be another factor that could also lead to the cooccurrence of HBsAg and anti-HBs in the chronic HBV
infection context. These antibodies could be directed against
HBsAg subtypes different from the coexisting HBsAg, justifying the serological profile [81, 83, 89]. In these cases,
HBV persistence in the presence of anti-HBs is not associated with the emergence of HBV escape mutants with
changed HBsAg sequences, but due to natural sequence
variations.
In such cases, it is possible that the major fraction of antiHBs antibodies in patients with concurrent HBsAg/anti-HBs
positivity may have the ‘wrong’ specificity and appear to be
unable to bind to serum HBsAg in the same patient. Importantly, it must be highlighted that the HBsAg sequence in
such cases demonstrates not the mutated HBsAg, but, rather,
the wild-type sequence associated with anti-HBs antibodies
that are heterotype specific. However, the mechanism by
which subtype-specific anti-HBs can be induced in patients
with chronic infection it is not clear.
Finally, the simultaneous detection of HBsAg and antiHBs in the bloodstream could be associated with a case of
viral reactivation (a phenomenon related to the presence of
occult infection), since the products of viral reactivation
could be a mutant, and the preexisting anti-HBs antibodies
could be targeted to wild-type virus [30].
Regarding the HBeAg in this serological profile, in all
situations addressed, its detection is likely to occur, since, in
the mechanisms described, there is no compromising of the
genomic sequence coding for the e protein. This means that
the HBeAg detection (or non-detection) in this context
would be more in dependence of the serological phase in
which the sample collection/analysis occurred (before or
after HBeAg clearance, in low- or high-replication phase),
than probably due to the production (or non-production) of
protein.
Profile 5
HBeAg negativity/positivity for anti-HBe and detectable
DNA
HBsAg [+]; HBeAg [−]; anti-HBe [+]; DNA/HBV [+]
It is universally acknowledged that HBeAg detection in the
circulation correlates with viral replication in the liver, while
its disappearance, associated with anti-HBe seroconversion,
is seen as a sign of replication absence and spontaneous
resolution of infection. However, this statement may not be
absolute, since, in the cases of precore region HBV mutants,
HBeAg expression may not occur, but there is maintenance
of viral replication, whose existence cannot be measured by
the HBe system [90]. In these cases, HBV/DNA and anti-HBe
antibodies can be found in the bloodstream at the same time,
justifying the occurrence of this atypical serological pattern.
The gene ‘C’ of HBV (nucleotide 1814 to nucleotide
2450) [82] is divided into the precore region and the core
region by two in-frame initiating ATG codons. Translation
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Eur J Clin Microbiol Infect Dis
from the second initiation codon (nucleotide 1901) results in
unprocessed core polypeptides (183-aa), which are assembled into HBcAg particles, and comprise the virion nucleocapsid. Initiation of translation at the first site (nucleotide
1814) produces a 312 amino acid polypeptide that has a
signal peptide directing it to the endoplasmic reticulum,
where the signal piece is removed by signal peptidases,
cleaving the N-terminal 19 amino acid residues. With subsequent processing, the C-terminal 34 amino acid residues
are removed, and the resultant polypeptide is secreted as
HBeAg, a soluble protein, detectable in the bloodstream [85,
90]. The function of secretory HBeAg in the viral life cycles
is unknown inasmuch as it is not required either for infection
or replication. However, it has been suggested that the
HBeAg may have a function in the immune response modulation during chronic HBV infection in adults and in promoting neonatal tolerance [15].
In the typical course of HBV infection or during antiviral
therapy in chronic HBV infection, the seroconversion of
HBeAg to anti-HBe is usually accompanied by a decrease
in viral replication and hepatic disease remission [90].
Among some patients, for unknown reasons, the immune
pressure associated with seroconversion contributes to the
viral variants emergence, which are characterized by not
expressing HBeAg or expressing at low levels, although
these patients have ongoing viral replication associated with
liver damage [90, 91].
During chronic HBV infection, two major types of HBV
core gene variants have been frequently reported, which
affect the HBeAg expression: the precore mutants and the
basal core promoter (BCP) mutants.
The most prevalent precore region mutation, known as
G1896A, is characterized by a guanine-to-adenine transition
at nucleotide position 1896, which converts codon 28 from
tryptophan (TGG) to a premature stop codon (TAG), and, by
so doing, aborts the translation of HBeAg precursor made of
29 amino acids coded for by the precore region and the 183
amino acids encoded by the core region [92]. The precursor
loses amino-terminal 19 amino acids and carboxy-terminal 34
amino acids to become HBeAg. This mutation occurs within
the epsilon (ε) structure, a highly conserved stem-loop at the
pregenomic RNA, essential for the initiation of encapsidation
and replication of the HBV pregenome, during the viral replication cycle [92]. However, the stability of this structure
depends on the strict conservation of base pairing in the stem
region, since mutations that alter the combination of these
bases may lead to less efficient viral replication or the production of non-viable viral particles [93].
Thus, in the ε structure, the base guanine, at the nucleotide 1896 (codon 28), on wild-type virus, forms a pair with
the base thymidine or cytosine of the nucleotide 1858 (codon 15), depending on the genotype involved [85]. In genotypes A, F, and H, the presence of cytosine at nt 1858
(C1858) stabilizes the ε structure and makes the occurrence
of a stop codon in the precore a rare event, maintaining the
preferred pairing G-C base proposed by Watson-Crick [65,
94]. However, the substitution of guanine by adenine at
position 1896 (A1896) would result in an unstable base
pairing (A-C), destabilizing the ε structure’s loop and also
reducing the efficiency of viral replication [65].
In contrast, the HBV genotypes B, D, and E, and some
strains of genotype C, have a thymidine at nucleotide 1858
(T1858). Thus, a mutation for adenine at position 1896
(A1896) stabilizes the ε structure, forming a new base pair
(A-T), in parallel, introducing a new stop codon [94] and
increasing the viral replication efficiency [65]. As a result,
viral strains which are HBeAg-negative are often found in
infections by genotypes B–E. With regard to genotype G,
the situation is peculiar, since all isolates are defined by the
presence of adenine at nt 1896, which introduces a stop
codon natural, making them incapable of expressing HBeAg
[94]. However, despite two stop codons (at positions 2 and
28) in the precore region characteristic of HBV genotype G
[92, 95], the HBeAg can still be detected in the serum of
infected individuals by this genotype, but this marker is
attributed to genotype A, with which the occurrence of coinfection is frequent [95].
In conclusion, patients infected with mutants in the precore region can then express high levels of DNA/HBV,
despite the presence of anti-HBeAg in the serum. However,
the cellular immune response modulation, induced by
HBeAg against the peptides HBeAg/HBcAg related and
expressed on the surface of infected hepatocytes, is lost
[96], which can correlate with exacerbation of liver injury
and a worse prognosis [97].
The second group of mutations affects the BCP (nt 1742
to nt 1849), with the double exchange of adenine by thymidine at nt 1762 (A1762T) and guanine by adenine at nt 1764
(G1764A) being the most common. These mutations are
often present in patients with chronic hepatitis or fulminant
hepatitis, and less frequently in asymptomatic carriers, in
immunosuppressed patients, and in infected individuals devoid of any serological markers [98]. They arise before or
during the HBeAg seroconversion to anti-HBe and result in
suppression at the transcriptional level of mRNA coding for
the core and precore. The consequence of these changes is
the decrease in HBeAg expression to approximately onethird of the levels observed in the wild viral strain and, also,
contribution to an improvement in the ability of viral genome replication [85, 90]. These observations are supported
by transfection studies which show low levels of preC
mRNA as well as of HBeAg secretion in the presence of
1762 T and 1764A [98].
As previously mentioned, it has been postulated that
HBeAg induces an immune tolerance against itself or to
HBcAg, or even against both antigens. As the HBeAg
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Eur J Clin Microbiol Infect Dis
shares some epitopes with the core antigen, its absence or
presence in low concentrations in the circulation causes the
HBcAg to be targeted directly by the humoral and cellular
immune system, leading to necrosis of hepatocytes and liver
damage [98]. This suggests a close relationship between
mutation in the BCP and liver disease advancement, which
leads to increased risk of developing hepatocellular carcinoma [99].
diminish the function of this transcriptional regulatory element [103], promoting the suppression of viral replication
and the synthesis of HBcAg and HBsAg. Consequently,
patients infected with X-defective HBV mutant are negative
for HBsAg and anti-HBc, despite the presence of HBV
replication and detectable DNA. X-defective HBV mutants
have been isolated from patients with acute or chronic
hepatitis [102].
Profile 6
Serological aspects of clinical and laboratorial
significance
Absence of serologic markers of infection and detectable
HBV/DNA
HBsAg [−]; HBeAg [−]; anti-HBc [−]; DNA/HBV [+]
The isolated detection of HBV/DNA in the absence of
serological markers is a typical serological/molecular profile
and a relatively common laboratory finding, particularly in
the blood transfusion context, when the research of this
molecular marker is already established in the donors
screening. At first, this finding does not seem curious or
unusual, since, in most cases, it can be observed progressively the emergence of other serological markers of viral
replication, which is indicative of infectious blood detection
still in a recent phase of infection.
However, the situations of persistently detectable DNA,
in the absence of other markers, can define an atypical
serological profile in HBV infection and lead to the suspicion of mutant virus infection, especially the X-defective
HBV mutant, whose characteristic is briefly described
below.
The X-ORF (X-region) encodes for a 154aa protein
called HBx (protein X). Protein X is known to transactivate
transcriptional regulatory elements, which include the enhancer and core promoter elements. The core promoter,
located at nt 1586 to nt 1849 [98, 100], regulates the transcription of the core/pregenome, and enhancer II, located at
nt 1687 to nt 1805 [101], is implicated in the activation of
the S-promoter [98, 101]. The sequence nt 1742 to nt 1849
is called the BCP. It is supposed to be sufficient for the
correct transcription initiation of the pregenome and precore
mRNA [82]. DR1 (direct repeated) and DR2, which are
implicated in the origin of HBV DNA, are also located in
the X-region.
The X-defective HBV mutant is commonly characterized
by an 8-nt deletion between nt 1770 and 1777 and a point
mutation of DR2 (T-to-C) in the X-ORF [102]. This deletion
results in a C-terminally truncated X protein of 134aa (due a
loss of 23aa and the addition of three normal aa), which
causes a loss of its transactivating activity. The 8-nt deletion
of the core promoter/enhancer II complex sequence may
Acute infection and non-detection of HBsAg
Classically, 2–3 months after contact with HBV—so still in
the incubation period, even before the biochemical evidence
of hepatic dysfunction—the detection of HBsAg is possible
[104]. Usually, it remains detectable in serum until about
2 months after the onset of clinical manifestations. However, this period may extend to over 3–6 months in individuals
with acute infection [1].
During this period (i.e., during 2 months after the onset of
clinical manifestations), the early clearance of HBsAg can
occur before marker identification is made in the laboratory.
This phenomenon is a result of the inhibition of HBV
replication (and decrease of HBsAg), whose mechanisms
are not well clarified, but are dependent on viral determinants (such as genotype, mutations) [105, 106], and also on
the host’s immune response, involving the induction of a
CD8(+) T-cell response (key cellular effectors mediating
HBV clearance from the liver) by CD4(+) cells (the master
regulators of the adaptive immune response to HBV), as was
demonstrated by Yang et al. [107]. Therefore, the negativity
of this marker does not exclude the diagnosis of acute HBV
infection!
Detection of IgM anti-HBc antibodies: acute or chronic
infection?
As already mentioned, IgM antibodies appear early, even
during the incubation period. When the disease is clinically
expressed, IgM and IgG antibodies are usually present.
After 2–6 months, IgM disappears, leaving only IgG antibodies [4].
In contrast to most viral infections, patients with acute
and chronic HBV often produce both IgM class and IgG
class anti-HBc antibodies; therefore, the mere presence of
IgM anti-HBc is not diagnostic of an acute infection. However, higher levels of IgM anti-HBc are generally produced
during the acute phase as compared with chronic infection,
and this quantitative difference can be the only serological
means of differentiating an acute HBV infection from an
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acute exacerbation of a chronic infection. Thus, the positivity for IgM anti-HBc antibodies should be analyzed with
caution so as not to induce errors in diagnosis. On the other
hand, in practice, anti-HBc IgM negativity is important,
because if this indicator is negative, the probability of acute
infection in HBsAg-positive cases is zero [104].
Correlation between anti-HBc and anti-HBe antibodies
production
Although HBcAg and HBeAg are serologically distinct,
they are structurally related, since these two proteins keep
in its structure a very extensive homology in the amino acid
residues sequence [53]. This has been observed from experiments that demonstrate the existence of cross-reactivity in
recognition of these antigens at the Th-cell level for antibodies production [28, 53], although the immune responses
to these antigens appear to be regulated independently.
It is known that anti-HBc antibody can be produced via
T-cell-independent and T-cell-dependent pathways, whereas
antibody production to the HBeAg is strictly T-celldependent [28]. Thus, the cross-reactivity of Th-cell recognition of HBcAg and HBeAg predicts a correlation between
the T-cell-dependent pathway of anti-HBc antibody production and anti-HBe antibody production. Accordingly, the
emergence of anti-HBe antibodies is associated with an
increasing T-cell response to the core protein, and is accompanied by a slight increase in anti-HBc antibodies [11].
Therefore, low anti-HBc titers may be consistent with only
T-cell-independent anti-HBc production, may reflect a deficit in HBe/HBcAg-specific Th-cell function, and are usually related to anti-HBe negativity in the serological profile.
On the other hand, high titers of anti-HBc antibodies are
associated with its production via T-cell-independent and Tcell-dependent pathways, and correlates with the presence
of anti-HBe antibodies in the serological profile [23], supporting the theory that, if T-cells assist in the anti-HBc
production, they would presumably also assist in the antiHBe production (Fig. 3). These data demonstrate that, although the anti-HBe antibodies presence is observed in only
33 % of anti-HBc-positive individuals [108], its detection
presupposes the existence of cellular immune mechanisms
responsible for HBV clearance, suggests convalescence, and
progresses toward recovery, whereas its absence in the profile may suggest the opposite. Obviously, the anti-HBe
detection in the absence of anti-HBc antibodies suggests a
profile initially incoherent and likely to be revised.
Seroconversion to anti-HBe and anti-HBs antibodies
The seroconversion to anti-HBe antibodies are frequently
correlated with viral clearance and remission of liver disease
during chronic infection. However, antibodies to HBeAg may
not develop in all HBV-infected patients, or may appear at
various times after the appearance of anti-HBc. The seroconversion to anti-HBs antibodies indicates viral elimination.
However, these antibodies can become undetectable in
patients who have recovered fully from infection. Although
there is still limited knowledge about the kinetics and regulation of HBs/HBe-specific antibody response [109], the low
HBs and HBe immunogenicity [110, 111] comprises one of
several potential immunological mechanisms that could explain the non-development or non-detection of these antibodies in the clinical course of HBV infection. Regardless of this
factor, it has been shown that antibody production to viral
protein HBe and HBs is a T-cell-dependent process [28], and
that the Th-cell response to HBcAg/HBeAg supports the
production of HBc and HBe antibodies, as well as antienvelope antibodies [112]. Considering that HBc/HBeAgspecific Th cells in humans mediate anti-envelope antibodies
production, and that there is a significant correlation between
the activation of CD4+ T lymphocytes by HBcAg/HBeAg
and the seroconversion phase to anti-HBe and anti-HBs in
acutely infected patients [11], it is tempting to postulate that
the seroconversion to anti-HBe antibodies predicts the seroconversion to anti-HBs antibodies. Nevertheless, it is possible
that anti-HBe antibodies can be detected even in the absence
of anti-HBs antibodies [113, 114], or that anti-HBs antibodies
are present alone in the serological profile (or in combination
with other markers, other than anti-HBe), without which these
serological situations are necessarily characterized as atypical
[114].
Prevalence of atypical serological profiles in HBV
infection
Atypical serological profiles in HBV infection include serological situations that tend to occur most frequently in the
chronic infection context in response to a long history of
HBV infection, associated with virological characteristics of
the agent (involving persistent viral replication, replication
mechanism, natural variability, and wide capacity of mutation), but also due to an inadequate HBV-specific immune
pressure of the host. And that is what the serological findings observed in studies conducted in regions of high prevalence of HBV infection (where the majority of infections
are contracted perinatally or in early childhood) have suggested, demonstrating that, in chronic carriers, involving
patients with chronic active hepatitis, or with chronic persistent hepatitis or asymptomatic carriers, the presence of
atypical patterns is more incident [86, 115, 116]. However,
these serological patterns can be observed in any clinical
setting of HBV infection, since its occurrence may also be
associated with the agent [117] and/or to the host [118], but
not just due to the long history of HBV infection.
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structural relationship
HBc antigen
HBe antigen
1
2
4
T-helper-cells
B-cells
HBe/HBcAg
specific
T-helper-cells [CD4+]
HBe/HBcAg specific
IgM anti-HBc antibodies
5
anti-HBc antibodies
anti-HBe antibodies
3
Detected months later
High immunogenicity
in accordance with the decrease
of IgM anti-HBc antibodies
[T-cell-independent and T-cell-dependent antigen]
[T-cell dependent antigen]
Correlation of the anti-HBe antibodies production and
anti-HBc antibodies via T-dependent
Fig. 3 Correlation of the anti-HBe antibodies production and anti-HBc
antibodies via T-cell-dependent pathways. 1 HBcAg directly stimulates
B cells to produce anti-HBc antibodies (primarily IgM); 2 HBcAg
requires T-cell helper function (which may be variably present from
patient to patient) in the production of IgG anti-HBc antibodies; 3
demonstrating its property to stimulate the anti-HBc antibodies
production via T-cell-independent as well as T-cell-dependent pathways (high immunogenicity); 4 HBeAg requires T-cell helper function
in the anti-HBe antibodies production; 5 emergence of anti-HBe antibodies (in correlation with high titers of anti-HBc) supported by the
cross-reactivity in recognition of these antigens at the T-helper-cell
level
Despite these conjectures, there are no data on the prevalence of these patterns (or which patterns are more prevalent) in the context of acute or chronic HBV infection, since
its occurrence, in most cases, have only been mentioned in
the context of other investigations on HBV [116, 119–121]
or identified primarily by routine laboratory tests in the
context of HBV diagnosis [122] (or accidentally, as a result
of screening protocols [123]), but not from serological studies correlating the incidence of atypical serological profiles
in a population of HBV-infected individuals.
deletions when compared with other genotypes [124].
Since deletions in preS/S has been a factor to justify the
co-occurrence of HBsAg and anti-HBs in the bloodstream
[79, 84], it seems plausible to associate a greater possibility
of detection of atypical serological patterns in the occurrence
of infection with this genotype [30].
However, it is observed that there is a poor relationship
between atypical patterns and genotypes of HBV, and, even
if there was such an association, the investigation of the
genotype would not comprise a tool of immediate practical
applicability to justify a particular atypical profile found,
especially in the context of serological routine diagnosis.
Even so, this correlation deserves further investigation.
Atypical serological profile and HBV genotypes
So far, there has been no study carried out to establish any
relationship between the presence of atypical patterns and
genotype of the infecting HBV. However, it seems logical to
assume that infection by certain genotypes, due to some
specific peculiarities, may justify this finding and even favor
its appearance. For example, with regard to infection by the
G genotype (possibly also by B and E), the occurrence/
presence of mutations in the precore region (or BCP) is
expected and may comprise a frequent finding in some
populations, especially in France and the USA, where infection by that genotype is incident [120]. Therefore, the
association of infection by this genotype with the coexistence of anti-HBe and DNA in the circulation, may be wide.
Another illustrative example is related to HBV/C genotype
infection. HBV/C has been given some peculiarities, among
which are the highest frequency of BCP mutations and preS/S
Conclusion
The natural course of hepatitis B virus (HBV) infection is
directly linked to immune tolerance, infection by viral variants, and viral clearance. The Immune tolerance, infection
by viral variants, and viral clearance, in turn, comprise
factors which are closely related to DNA levels produced
by the agent, genetic expression of viral antigens, and also
the patient’s immune response. The interrelationship of
these factors, most of the time, may justify the identification
of atypical serological patterns which are beyond those
classically known and universally accepted, such as those
described here. Thus, it seems possible that acute or chronic
carriers of HBV are identified, showing serological patterns
that do not characterize them as such.
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Moreover, the advent of the next generation of immunoassays, the improvement in the sensitivity and specificity of
molecular assays, and better knowledge of the biology of
HBV also tend to reveal other curious serological situations,
such as HBsAg detection in the absence of HBV/DNA
[125], and which will be added to a constellation of atypical
profiles, which are yet to be revealed, in several clinical
contexts of HBV infection. These profiles could be a problem in the medical clinic and laboratory diagnosis of hepatitis B, but, also, could be able to characterize different
infection phases and even revise the current clinical concepts of HBV infection.
Conflict of interest
conflict of interest.
The author has declared that they have no
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