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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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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 response, and, therefore, clinical practice diagnosis of
four antigen–antibody systems are observed: HBsAg and HBV infection is established by the serological detection
anti-HBs; preS antigen and anti-preS antibody; HBcAg of HBV protein products (antigens) as well as host-
and anti-HBc; and HBeAg and anti-HBe. Through the ex- produced antibodies. During HBV infection, four structural
amination of these antigen–antibody systems, hepatitis B antigen–antibody systems are observed: hepatitis B surface
infection is diagnosed and the course of the disorder may antigen (HBsAg) and its antibody (anti-HBs); the preS anti-
be observed. Although the serologic findings that allow both gens associated with HBsAg particles and their antibodies;
the diagnosis of HBV infection as well as assessing of its the particulate nucleocapsid antigen (HBcAg) and anti-HBc;
clinical course are already well established, the dynamics of and an antigen structurally related to HBcAg, namely, hep-
viral proteins expression and of the antibodies production atitis B e antigen (HBeAg) and its antibody (anti-HBe).
may vary during the infection natural course. This causes the Through the examination of these antigen–antibody sys-
HBV infection to be occasionally associated with the presence tems, hepatitis B infection is diagnosed and the course of
of uncommon serological profiles, which could lead to doubts the disorder may be observed
in the interpretation of results or suspicion of a serological Classically, 8–9 weeks following the infection, in the
result being incorrect. This paper is dedicated to the discussion incubation period or 3–5 weeks before biochemical evi-
of some of these profiles and their significance. dence 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
Introduction and indicates potential infectiousness. In patients who sub-
sequently recover from HBV infection, HBsAg usually
The natural course of hepatitis B virus (HBV) infection is becomes undetectable after 4–6 months [1]. If HBsAg per-
determined by the inter-relationship between viral replica- sists for more than 6 months, spontaneous clearance is very
tion via HBV protein production and the host’s immune improbable and the infected individual is considered to be a
chronic HBV carrier [2].
R. A. A. Pondé However, during the acute phase of infection, following
Laboratório de Virologia Humana, Instituto de Patologia HBsAg detection, other viral markers can be easily detect-
Tropical e Saúde Pública, Universidade Federal de Goiás,
able, including DNA polymerase and HBeAg. HBeAg
Goiânia-Goiás, Brazil
appears shortly after the appearance of HBsAg and disap-
R. A. A. Pondé pears within several weeks as acute hepatitis resolves. Its
Gerência de Hepatites Virais, presence in the serum correlates with the presence of viral
SUVISA - Superintendência de Vigilância em Saúde - Secretaria
replication in the liver and HBsAg detection, while its
Estadual de Saúde, Goiânia-Go, Brazil
disappearance, associated with the anti-HBe detection, is
R. A. A. Pondé (*) seen as a sign of the absence of viral replication and spon-
Sorologia e Biologia Molecular, Central Goiana de Sorologia, taneous resolution of acute infection.
Rua 7A Edifício RIOL,
Anti-HBc IgM antibodies are detectable at the outset of
N° 158, 1° andar, sala 101, Setor aeroporto,
Goiânia-Goiás 74075-030, Brazil clinical disease, and, as the infection evolves, IgM anti-HBc
e-mail: roberioponde@uol.com.br levels gradually decline, often becoming undetectable
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Eur J Clin Microbiol Infect Dis

within 6 months and IgG class predominates, remaining for to diagnose the presence of infection and its development,
a long period of time (sometimes life-long) at detectable according to the dynamics described above. Although the
levels [2]. The presence of IgM anti-HBc usually indicates serologic findings that allow both the diagnosis of HBV
recent or continuous HBV replication, while the presence of infection as well as assessing its clinical course are already
IgG anti-HBc correlates with past infection [3]. well established, the dynamics of viral proteins expression
As recovery and convalescence signal, antibodies to oth- and of the antibodies production may vary during the infec-
er viral proteins appear in the blood. Anti-HBe antibodies tion natural course, due to factors related to the agent (in-
appear after HBeAg clearance, and may persist for many fection by a new strain or viral serotype—antigenic variant)
years after the resolution of acute HBV infection. In some and/or to the host (immune tolerance, cellular immune re-
cases, the phenomenon of “immunological window” of the sponse, and immunosuppression) [5–7]. This causes the
HBe-anti-HBe system can be observed, with anti-HBe anti- HBV infection to be occasionally associated with the pres-
body detection about 2 months after the disappearance of its ence of uncommon serological profiles, which can lead to
antigen precursor. doubts in the interpretation of results or suspicion of an error
The antibody response to hepatitis B surface antigen (anti- in the serological diagnosis. The purpose of this paper is to
HBs) becomes detectable in convalescence, from 6 weeks to discuss the atypical profiles most frequently found in HBV
6 months after HBsAg clearance, although it is produced early serology.
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 Profile 1
HBsAg and infectious HBV particles from peripheral blood.
These neutralizing antibodies are especially important in the Isolated positivity for HBsAg and detectable DNA
prevention of viral infection, since they could prevent viral
attachment and entry into the cells by absorption of the viral HBsAg [+]; HBeAg [+/−]; anti-HBc [−]; anti-HBe [−];
particles [3]. Its presence is considered to be an indicator of anti-HBs [−]; DNA/HBV [+]
immunity to HBV infection, although it could become unde-
tectable in patients who have recovered fully from infection The isolated positivity for HBsAg comprises a finding often
(for a review, see [4]). Table 1 shows the dynamics of the observed in HBV serology whose ample meaning was dis-
expression of HBV serological markers, the corresponding cussed recently [8]. Accordingly, this serological profile, as-
antibodies, and the interpretation of the profile, in accordance sociated with DNA detection in the bloodstream, suggests,
with the evolution in an typical serological profile, during the initially, acute infection, which is a consequence of recent
natural course of infection. exposure to HBV, and this hypothesis, in most cases, can be
Currently, the most accurate tool available for the diag- confirmed after the subsequent expression of other markers
nosis of HBV infection is DNA detection, usually obtained detected by laboratory testing. However, the persistent isolat-
by genomic amplification assays. However, anti-HBV- ed positivity for HBsAg and detectable DNA, in the absence
specific tests are used routinely: (i) to assess disease activity of other markers, comprises an atypical serological profile,
in persistent infection; (ii) in monitoring therapeutic regi- whose presence may indicate immune tolerance to HBV core
mens with antiviral agents, and, especially, (iii) in an attempt 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 NEG NEG NEG NEG NEG Incubation period


POS POS POS POS NEG NEG Acute infection
POS NEG NEG POS NEG NEG HBV infection (acute or chronic HBV infection)
POS NEG NEG POS NEG/POS NEG Chronic HBV infection or end of recent infection
NEG NEG POS POS NEG NEG Recent HBV infection. Beginning of the convalescence period
NEG NEG NEG POS NEG NEG Past HBV infection (immunological window)
NEG NEG NEG POS POS POS Immune, recent past infection
NEG NEG NEG POS NEG POS Immune, past infection (old infection)
NEG NEG NEG NEG NEG POS Immune, contact HBsAg, vaccine response
NEG NEG NEG NEG NEG NEG Susceptible individual. Never had contact with HBV
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suggest infection by an HBV mutant, factors that can com- other hand, following the occurrence of core gene deletions,
promise anti-HBc antibodies production. depending on the region involved, the HBeAg expression
The immune tolerance to HBcAg is known by the incapac- may become compromised, since both antigens are encoded
ity of the individual to produce anti-HBc or to produce it in by the same gene.
undetectable levels. The immune tolerance can be mediated Additionally, it is of great interest, and deserves high-
by a variety of mechanisms. For example, T-cell anergy is a light, the observation of this serological profile in the chron-
tolerance mechanism in which the T cell is functionally inac- ic infection context. Serologically, the chronic HBV
tivated following an initial antigen encounter, but remains infection is characterized by persistence of HBsAg in the
alive in a hypoactivated state [9]. T-cell anergy has been bloodstream for more than 6 months, associated with the
proposed as a major mechanism for the maintenance of self- presence of anti-HBc antibodies and detectable DNA. Dur-
tolerance and regulation of the cellular immune response [10]. ing the natural course of chronic infection, the spontaneous
In addition, a small number of specific peripheral T lympho- seroclearance of HBsAg and even of DNA can occur after
cytes, an inefficient antigen presentation, or lymphokine pro- antiviral therapy, but anti-HBc antibodies remain detectable
duction by the antigen-presenting cell are, among others, continually in peripheral blood [21]. However, in some
possibilities associated with anti-HBc production [11]. There- circumstances, the presence of anti-HBc antibodies may
fore, a selective immune system defect could lead the immune not be observed in this clinical context, but only isolated
tolerance to the HBc and, consequently, the lack of production HBsAg positivity and DNA detectable in the serological
of corresponding antibody, justifying, therefore, the existence profile, comprising an unusual finding in medical practice.
of this serological profile [12, 13]. In this case, the serological pattern is justified by immune
The phenomenon of the immune tolerance to HBc anti- complexes formation of anti-HBc antibodies with an excess of
gen has also been associated with the vertical transmission HBc antigen in the bloodstream, with anti-HBc becoming
of infection, and, consequently, the absence of an antibody undetectable [22]. These immune complexes cannot be
response to HBc in infants born to HBe-positive carrier detected by available commercial tests, which are not able to
mothers [14]. It has been postulated that HBeAg may have detect antibodies in the presence of excess antigen in the serum
an immunoregulatory function in promoting viral persis- [23]. Anti-HBc complexes could be detected only after immune
tence [15]. The HBcAg and HBeAg are distinctly recog- complex dissociative treatment. Some methods are based on
nized by antibodies but, due to their extensive amino acid polyethylene glycol precipitation followed by ion chaotropic
homology, are highly cross-reactive at the T-cell level [15]. treatment [24] or based on sucrose gradient fractionation [25].
Thus, HBe antigen, because of its small size, may traverse HBcAg is not a secreted protein and exists primarily in
the placenta and elicit HBe/HBcAg-specific T-helper cell liver and in serum within HBV particles, not being directly
tolerance in utero [16]. Consequently, it may lead to fetal accessible in the blood to the immune system. In exceptional
immunotolerance not only to HBeAg but also to HBcAg, cases, HBc epitopes may, however, be exposed to virions
and still inhibit anti-HBc antibody production when it is present in the serum [26] and also during liver necrosis,
present in the serum. when nucleocapsids may be secreted from infected hepato-
As mentioned earlier, it is possible that agent-related cytes with a high level of HBV particles, explaining the
factors can also justify the anti-HBc non-production by the presence of anti-HBc complexes in the bloodstream. This
host and contribute to the expression of this serological phenomenon has been observed in chronic HBV carriers
profile. Among them include infection by HBV which pro- during the phase of active replication in a state of immuno-
duces a non-responsive HBc [17, 18], therefore, unable to suppression, as seen in the context of transplantation (bone
induce an immune response, or by HBV mutant with nucle- marrow, kidney, heart), during chemotherapeutical treatment
otide sequence deletions in the core gene. These deletions for neoplasias [27], or an uncontrolled human immunodefi-
may involve important epitopes to antigen recognition by ciency virus (HIV) infection, conditions which could also
immune response cells, and, therefore, compromise the HBc lead to an immune system defect. HBcAg is the most
antigenicity, becoming a non-functional protein [19, 20]. immunogenic HBV component and induces the production
The presence or absence of HBeAg in this profile may be of high titers of anti-HBc antibodies in immunocompetent
inconsistent or even vary from case to case, since its detec- individuals who have been exposed to HBV [28]. However,
tion (or not) will be associated with the mechanism that the immunosuppression conditions can potentially induce
justifies the profile in question. For example, in the case of decreased antibody production [3]. HBcAg-reactive materi-
immune tolerance specific to c antigen or due to vertical al is released from HBV particles present at a high level in
transmission [12, 13], the HBeAg detection in the profile it the serum and from hepatocytes undergoing lysis. Conse-
seems likely, since, in these cases, there is no compromising quently, this could complex to low levels of anti-HBc pro-
of the viral antigens expression (the preC/C regions remain duced in these patients and, therefore, lead to the non-
unchanged, with normal synthesis of viral antigen). On the detection of antibodies (Fig. 1).
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Phase of active
Chronic HBV carrier Hepatocyte lysis Immune-complex [IC] Immunosuppression
replication [HBcAg-anti-HBc] [anti-HBc in low levels]

HBsAg [+] / HBV free HBcAg free Anti-HBc [+] [not complexed] Anti-HBc [ - ] and HBsAg [+]
Anti-HBc [+] [free] / HBeAg [+/-] [in the serum] HBcAg complexed HBcAg [free] / Anti-HBc-HBcAg [IC]

Anti-HBc antibodies HBV HBc Antigen Anti-HBc-HBcAg [IC]


HBsAg

Fig. 1 Schematic representation of isolated positivity for HBsAg in subsequent complexation with anti-HBc free in excess. In the immu-
the chronic hepatitis B virus (HBV) infection context, due to the nosuppression conditions (and low levels of anti-HBc antibodies), after
formation of immune complexes HBcAg and anti-HBc. In chronic hepatocytes lysis, the HBcAg released (in excess) complex with anti-
HBV carriers, during the phase of active replication, hepatocyte lysis HBc antibodies, leading to the non-detection of antibodies and isolated
occurs, as well as the release of HBcAg in the bloodstream, and 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
Positivity for anti-HBc antibodies, with or without detection cases and the non-detection of HBsAg in this serological/
of anti-HBs antibodies and detectable DNA molecular context.
The molecular basis of the occult infection seems to be
HBsAg [−]; anti-HBc [+]; anti-HBs [+/−]; DNA/HBV [+] strictly related to the peculiar life cycle of HBV. In particu-
lar, a fundamental step is the conversion of the relaxed
Against the classical information that the anti-HBc detection circular genome into a covalently closed circular DNA
in association with anti-HBs antibodies in the bloodstream (cccDNA). This comprises a long HBV replicative interme-
is indicative of viral clearance and immunity, this serologic/ diate that persists in the cell nuclei (hepatocyte) as a stable
molecular profile is indicative of persistent infection and has episome and that serve as the template for mRNAs and
been associated with the presence of occult HBV infection. RNA pregenomic transcription [34]. During the viral repli-
The anti-HBc antibodies detection is justified by the high cation cycle, viral transcript is translated in capside, together
immunogenicity of the core antigen [29] and includes a with polymerase proteins. After subsequent encapsidation,
classic finding in cases of exposure to HBV. On the other RNA pregenomic is retranscripted into new partial double-
hand, the anti-HBs antibodies (when present), in the context strand viral genomes. The new nuclear capside containing
of this serological/molecular profile, can be of heterologous DNA may be secreted as a complete virion after the attach-
nature (i.e., directed to another HBsAg antigenic determi- ment of coating protein or, then, may redirect the viral
nant) or be directed to HBsAg (mutant or not, but not genome to the hepatocyte nucleus to pool the maintenance
detectable) of the first viruses infecting, which does not of cccDNA and to retain permanently infected cells [31, 35,
recognize, however, the persistent HBV, not having, there- 36]. Thus, considering the stability and long-term persis-
fore, any protective or neutralizing activity of the infection tence of viral cccDNA molecules in the liver tissue, it seems
developing [30]. The generation mechanism of anti-HBs plausible to affirm that, (i) HBV infection, once it has
antibodies and its significance will be discussed in detail occurred, may possibly continue for life, and (ii) resolution
in “Profile 4”. This discusses the non-detection of HBsAg in of disease does not imply complete eradication of infection,
the context of occult infection, justifying the serological/ but, more likely, reflects the capacity of the host’s immune
molecular profile in question. response to restrain HBV and to keep persistent control of its
Occult infection can be defined as the long-lasting per- replication [33, 35].
sistence of viral genomes in the liver tissue (and, in some Accordingly, histopathological analysis of the liver by
cases, also in the serum) of individuals negative for the immunohistochemistry in individuals with occult infection
HBV surface antigen [31]. In many instances, occult hepa- has revealed the expression of HBsAg or HBcAg in the liver
titis B is associated with hepatitis B core antibody (anti- tissue comparable to those of chronic HBV carriers, despite
HBc) and/or anti-HBs [32]. In this context, the viral genome the signals being lower [37, 38]. Additionally, the presence
persistence under conditions not yet elucidated is character- of extrachromosomal HBV/DNA and pregenomic RNA in
ized by a strong suppression of viral replication and expres- the liver and peripheral blood mononuclear cells by molec-
sion of its genes, identified only by highly sensitive ular techniques [polymerase chain reaction (PCR) and in
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situ hybridization] has been demonstrated in some patients, detect an antibody in the form of immune complexes [23].
suggesting the maintenance of transcriptional activity, and, Another explanation for failure in detecting anti-HBs could
therefore, HBV activity [39]. Finally, in the chronic infec- be the low sensitivity of commercial assays currently stan-
tion context, the persistence of replicative forms of the viral dardized, which are not adjusted to detect antibodies pro-
DNA in the hepatocytes nucleus, after HBsAg disappear- duced in low levels, since an increase in its sensitivity would
ance (spontaneously or due to a successful treatment), has also increase the risk of false-positive results [48, 49]. This
also been detected by in situ hybridization, despite the observation suggests that the marker detection depends on
inhibition of DNA synthesis by antiviral therapy [40]. These the sensitivity of the test adopted and of the detection limit,
data illustrate that, (i) it is possible that some patients, even which is arbitrarily determined by the manufacturer [49, 50].
after the loss of serological markers of active infection, may Finally, the occurrence of residues substitutions in
still demonstrate a low degree of infection with liver disease HBsAg, involving specific epitopes, could be able to pre-
development, despite the antibodies presence, and (ii) the vent the protein recognition by T-cells CD4+ or minimize
persistent infection can occur without the production of viral the ability of its stimulation, compromising, consequently,
particles. The frequency with which this occurs is unknown, the anti-HBs production [51, 52] and its detection in the
although there are reports which have indicated that 3 out of serological profile.
every 16 monitored patients after acute infection remain In conclusion, in this clinical setting, the detection or not
viremic for a period of several years [41, 42]. of anti-HBs antibodies in the serologic profile HBsAg [−];
Even considering the low levels of replication and intra- anti-HBc [+]; anti-HBs [+/−]; DNA/HBV [+], does not
hepatic HBV transcription maintained during the natural seem to have great significance and implications, since these
history of occult infection in these individuals, a question antibodies, in detectable levels, do not fulfill its function of
remains open. What reasons would justify the non- neutralizing, whereas its presence below detection levels,
detection/expression of HBsAg in this situation? Two pos- despite characterizing the presence of immune response, is
sibilities have been discussed: the antigen may be absent insufficient to prevent viral replication and establish infec-
from the peripheral blood or may be present but not detect- tion control.
able. 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 Profile 3
which would block HBsAg secretion, causing its accumu-
lation inside the cell [38, 44]. In the second situation, the HBeAg positivity/negativity for HBsAg
HBsAg may be present but, however, escaping, the recog-
nition by enzyme immunoassays currently standardized, HBsAg [−]; HBeAg [+]; anti-HBc [+]; DNA/HBV [+]
depending on the sensitivity of detection [45]. There is,
however, an additional explanation for the non-detection HBeAg is a secretory protein that is processed from the
of HBsAg: mutations in regions that code for protein S precore protein of HBV. It is generally considered to be a
[46]. Specifically, mutations in the major hydrophilic region marker of HBV replication and infectivity, although it is not
of HBsAg, the main target for antibodies used in diagnostic required for infectivity or viral replication [53]. As a non-
tests. These mutations could lead not only to an escape from structural component of HBV, HBeAg is abundantly secret-
recognition by the tests routinely used, but may also reduce ed into the bloodstream during the acute phase of infection,
the replicative capacity of the virus, reducing the levels of appearing shortly after the appearance of HBsAg. Classical-
HBsAg, which could compromise its detection in the blood- ly, the HBeAg presence in the bloodstream correlates with
stream. Bréchot et al. [47], however, have provided strong high levels of viral replication in hepatic tissue and HBsAg
evidence that most cases of occult HBV is more related to detection in serum. Therefore, HBeAg positivity/HBsAg
low levels of HBV wild-type (low viral load) than the negativity in the serological profile comprises an atypical
presence of HBV mutants that do not express surface pro- pattern that deserves to be addressed.
teins or that produce with conformational structure changes. The ‘a’ determinant of HBV consists of a defined sero-
Concerning the absence of anti-HBs antibodies in the logical region, located between amino acid residues 124 and
serological profile, the failure to detect these antibodies in 147 of the HBsAg, which induces a protective immune
many patients in this context does not necessarily indicate response common to all HBV subtypes [54]. Variations in
the absence of humoral immune response, and may be its primary structure have been demonstrated to markedly
justified by the formation of immune complex HBsAg alter the antigenic conformation and antigenicity of HBsAg.
anti-HBs. As a practical proof of this fact, it has been shown For example, the loss of the proline at position 120 signif-
that anti-HBs can be detected in all the patients that evolve icantly minimizes the binding of antibodies mapped to that
into chronic infection, when measured by an assay able to region, as does the insertions between residues 122 and 124
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[55]. A similar effect is the occurrence of substitutions at Profile 4


positions 122 and 123, which demonstrate that this region is
a critical site in the HBsAg antigenicity [56]. In vitro studies Simultaneous positivity for HBsAg and anti-HBs
have shown that the HBsAg antigenicity is also lost when (and detectable DNA)
cysteine residues at positions 124, 137, 139, 147, or 149 are
replaced by serine, suggesting that this amino acid is of HBsAg [+]; HBeAg [+/−]; anti-HBc [+]; anti-HBs [+];
fundamental importance in maintaining the conformational DNA/HBV [+]
structure of the molecule [57]. Thus, the maintenance of the
tertiary molecular structure of HBsAg, especially of its Concurrent HBsAg and hepatitis B surface antibodies com-
immunodominant region, is of crucial importance to recog- prise a peculiar serological finding, since it contemplates
nition by antibodies, since antibodies directed to this region components that indicate immunity and, at the same time,
protect against HBV infection, and HBV variants that are developing infection. However, despite the presence of neu-
not recognized by them can escape its protective action [58]. tralizing antibodies, this serological profile correlates with
On the other hand, the commercial tests usually detect active viral replication and may be observed in acute or
this protein using a capture monoclonal antibody and a chronic hepatitis B infection, although the mechanisms that
detection monoclonal antibody (or polyclonal) anti-HBs could lead to this serological pattern have not been well
wild-type, such as those produced after vaccination [45, delineated and understood. Its existence has been justified
46]. The antibodies bound to the antigen captured tend to mainly by the emergence of HBV mutants and the presence
be specific for epitopes present in the determinant ‘a’ of of non-protective anti-HBs antibodies, as was recently dis-
HBsAg and oriented to recognize its cyclic peptide se- cussed [30].
quence [45, 54]. Since the immune response to HBsAg is In the acute infection context, the identification of this
primarily directed against conformational epitopes of the ‘a’ profile may be justified by the occurrence of reinfection
determinant, and that its antigenicity has been employed for with a new HBV strain. This new HBV strain could have
HBsAg detection in the serum [54, 59], changes in its an ‘a’ determinant different from the first HBV infection
tertiary structure could not only prevent the binding of (mutant), whose circulating antibodies would not have a
neutralizing antibodies targeted to the wild-type virus, but, neutralizing action, and, therefore, were not protective. Ob-
also, the antigenic detection by the assay used for diagnosis viously, in this case, the presence of anti-HBs does not
[58, 60, 61] (depending on the epitopes recognized by the indicate the removal of HBV and means that HBV carriers
test configuration [62]), leading to false-negative results, with concurrent anti-HBs still have active virus replication,
justifying, therefore, the serological profile HBeAg [+]/ suggesting that some of the infecting viruses are surface
HBsAg [−] and detectable DNA. antigen mutants.
Recent studies have shown conflicting results between The emergence of HBV escape mutations has been a
the tests applied in the detection of HBsAg mutants [63, 64]. phenomenon associated with the replication mechanism
This has occurred, presumably, due to differences existing (see below), and also as a result of a selection process due
between the tests applied, related to the analytical sensitivity to a selective advantage of variants during the course of
(> or <0.1 ng/ml) or associated with the system configura- HBV infection in a patient (for a review, see [6, 7, 66]).
tion of capture and detection. With regards to the capture Additionally, the primary prevention of HBV infection, such
and detection system, the sensitivity of the tests currently as vaccination in infants born to HBsAg-positive mothers
designed to detect HBV mutants can be variable, being and immunoprophylaxis in orthotopic liver transplantation
established between 57 % for testing monoclonal/capture recipients, due to exerting strong selective immune pressure
and 80.6 to 97.4 % for testing polyclonal/capture, which has on HBV, in some cases, can also induce escape mutations
been linked to the non-recognition of HBsAg epitopes by within the ‘a’ determinant region, affecting several amino
monoclonal anti-HBs antibody used in the test [65] and acid positions, as previously mentioned [67–70]. HBV
shows a significant advantage of polyclonal capture sys- mutants can be extremely stable and maintain their ability
tems. However, it is known that the polyclonal tests do not to replicate at high titer in the presence of anti-HBs [67].
guarantee full sensitivity in the HBsAg mutant detection, They can also be transmitted horizontally to other
which makes it important to incorporate specific antibod- humans, and, therefore, justify cases of HBV reinfection/
ies against mutant forms of HBV in commercial tests. superinfection [71]. HBV reinfection comprises a common
Thus, it should be noted that the test configuration is phenomenon in the liver transplantation context, reaching
not the only factor that predicts the capability to detect rates of more than 90 % if there is no treatment [72]. With
the mutant, but, rather, the ability of epitope recognition the use of LMV and HBIG for prophylaxis, these rates
by the reagent used to immobilize or detect the antigen can be reduced to almost 3.0 %, as demonstrated by Wang
involved [64]. et al. [73].
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Cases of HBV infection in previously vaccinated individ- HBV produces three envelope proteins which are all
uals may also occur [74, 75], and justify the co-existence of encoded in the preS/S open reading frame. The major hep-
HBsAg/anti-HBs serological profile. Obviously, this would atitis B surface (HBsAg) is coded by the S gene. The middle
not be a case of HBV reinfection (since the anti-HBs present HBsAg is coded by the preS2 and S genes. The large
in the bloodstream was induced by vaccination), but dem- HBsAg is coded by the preS1, preS2, and S genes. The
onstrates the possibility of replication of HBV (which pro- preS sequences exhibit the highest heterogeneity of the
duces an HBsAg mutant or not, but which is different from HBV genome [84, 85]. Mutations in the preS region, such
HBsAg vaccine), in the presence of preexisting anti-HBs as deletions involving the preS2 translation initiation codon,
antibodies (vaccine-induced), but which is unable to recog- could promote the synthesis of an anomalous HBsAg de-
nize the circulating virus (Fig. 2a). tectable in the bloodstream, but, however, not recognized by
In the chronic infection context, the observation of this previously anti-HBs antibodies formed against the original
profile can be justified by the detection of HBV mutants HBsAg [76, 86]. These mutations can arise in the natural
naturally selected due to existing anti-HBs endogenous re- course of HBV infection and have been more frequently
sponse [76–79]. Consequently, some chronic carriers have observed in patients with anti-HBs who did not receive
HBsAg e anti-HBs; however, the antibody detected is being hepatitis B immunoglobulin or vaccine than in those HBV
directed against HBsAg epitopes not shared by original carriers who do not have anti-HBs. These mutations are
HBsAg present in the bloodstream, and, therefore, not pro- more frequently observed in asymptomatic carriers [86].
tective [80] (Fig. 2b). In this case, the presence of anti-HBs Deletion in the preS1 region has also been associated to
does not indicate viral elimination and suggests that HBV the co-occurrence of HBsAg/anti-HBs, being related with
carriers with HBsAg and anti-HBs demonstrate active rep- persistent viremia. In a previous study, a deletion of nucle-
lication. Moreover, the presence of anti-HBs does not ex- otide 31 of the HBs gene in a patient with anti-HBs sero-
clude the possibility of a new HBV infection or the conversion but who still remained an HBV carrier with a
reactivation of preexisting HBV infection. virus load of 104 DNA molecules/ml of serum was ob-
These mutants have been characterized, presenting dele- served. This deletion led to frame-shift and introduced a
tions [80, 81], point mutations [82], and genetic recombina- stop codon after the 21 amino of HBs. The resulting HBsAg
tion [83] within the preS/S genes, which have been lacking the major epitopes recognized by specific antibodies
identified, frequently, in HBV DNA sequences obtained could favor ongoing viral replication, despite the presence
from the sera of inactive carriers. of anti-HBs [81].

Fig. 2 a Schematic
representation of a infection
a
with a new HBV strain in a Vaccinee individual (immune) Infection [New HBV strain] Acute infection/viremia
vaccinated individual
(immune), demonstrating non-
recognition and non-
neutralizing of the circulating
virus by anti-HBs antibodies
induced by vaccination. b Anti-HBs antibodies [+] HBsAg [+] / HBV free HBsAg [+] [New HBV strain]
Schematic representation
[vaccine-induced] [unrecognized] Anti-HBs [+]
showing emergence of HBV
mutants induced by immune
pressure (anti-HBs endoge- Anti-HBs antibodies New HBV strain New HBsAg
nous). In the chronic infection
context, the HBV/HBsAg exists
in both forms, free and
b
Immune pressure
immunoglobulin-bound,
whereas the anti-HBs antibod- Chronic Infection Mutation Seroconversion
ies only exist in the form of
immune complex. Due to the
immune pressure (anti-HBs in-
duced), the emergence of HBV
mutants with later HBV sero-
conversion (to wild-type virus) HBV/HBsAg [+] free HBV/HBsAg [+] mutant Anti-HBs [+] free [detectable]
and consequent simultaneous
Anti-HBs [+] not detectable (free) HBsAg [+] mutant [free]
detection of anti-HBs and
HBsAg mutant in the blood-
stream occurs HBV HBV/HBsAg immunoglobulin-bound HBV Mutant Anti-HBs free
HBsAg HBsAg Mutant [anti-wild type]
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Eur J Clin Microbiol Infect Dis

Besides deletions, and as aforementioned, variations in HBV persistence in the presence of anti-HBs is not associ-
the primary structure of the ‘a’ determinant have also con- ated with the emergence of HBV escape mutants with
tributed to justifying the presence of this profile, since these changed HBsAg sequences, but due to natural sequence
variations may markedly alter the antigenic conformation variations.
and antigenicity of HBsAg. Therefore, the binding of both In such cases, it is possible that the major fraction of anti-
monoclonal and polyclonal anti-HBs antibodies can be af- HBs antibodies in patients with concurrent HBsAg/anti-HBs
fected by a single aa change, and antibody raised to the positivity may have the ‘wrong’ specificity and appear to be
wild-type can be bound less well to the HBsAg variant. On unable to bind to serum HBsAg in the same patient. Impor-
the occurrence of these variations, there could be, then, the tantly, it must be highlighted that the HBsAg sequence in
presence of HBsAg mutant and the concomitant presence of such cases demonstrates not the mutated HBsAg, but, rather,
anti-HBs antibodies not bound to the mutated antigen. the wild-type sequence associated with anti-HBs antibodies
It must be highlighted also that an additional cause to that are heterotype specific. However, the mechanism by
justify the mutants emergence during the natural course of which subtype-specific anti-HBs can be induced in patients
HBV infection has been linked to the complex mechanism with chronic infection it is not clear.
of viral replication, characterized by the synthesis of viral Finally, the simultaneous detection of HBsAg and anti-
DNA through an RNA intermediary, by reverse transcrip- HBs in the bloodstream could be associated with a case of
tion [87]. As result of this mechanism and due to biochem- viral reactivation (a phenomenon related to the presence of
ical processes of the host cell (besides the infidelity of the occult infection), since the products of viral reactivation
viral replication machinery), errors of bases incorporation could be a mutant, and the preexisting anti-HBs antibodies
into DNA strands may inevitably occur during viral repli- could be targeted to wild-type virus [30].
cation. Considering the large number of viral particles gen- Regarding the HBeAg in this serological profile, in all
erated in an infected person (1011 viral particles per day), situations addressed, its detection is likely to occur, since, in
and that the error rate (because of the polymerase reverse the mechanisms described, there is no compromising of the
transcription) can be of the order of 1 error per 1010 bases, in genomic sequence coding for the e protein. This means that
active infection, 1010 base-pairing errors could be generated the HBeAg detection (or non-detection) in this context
per day over the 3,200-bp genome in a given patient [88]. would be more in dependence of the serological phase in
These errors lead to the generation of multiple variant tran- which the sample collection/analysis occurred (before or
scripts from a single template, and, consequently, the for- after HBeAg clearance, in low- or high-replication phase),
mation of a quasispecies pool [88]. While these new than probably due to the production (or non-production) of
sequences are viable, they effectively compete with the wild protein.
type and comprise a material source for the emergence of
mutants in an environment with significant selective im-
mune pressure during the natural course of HBV infection. Profile 5
In this context, in the proportion that any newly generated
mutation confers selective advantages to the virus, it could HBeAg negativity/positivity for anti-HBe and detectable
allow the corresponding viral population to express more DNA
than wild-type virus, indirectly contributing to HBV
seroconversion. HBsAg [+]; HBeAg [−]; anti-HBe [+]; DNA/HBV [+]
Thus, HBsAg/anti-HBs simultaneous detection could oc-
cur, suggesting that the acquisition of anti-HBs could be It is universally acknowledged that HBeAg detection in the
associated with the emergence of immune escape variants circulation correlates with viral replication in the liver, while
persisting for several years in association with wild-type its disappearance, associated with anti-HBe seroconversion,
strains. The presence of HBsAg variants would arise naturally is seen as a sign of replication absence and spontaneous
in chronic carriers and should be a viral strategy to evade the resolution of infection. However, this statement may not be
immune system. Therefore, the detectable HBsAg is a mixture absolute, since, in the cases of precore region HBV mutants,
of the wild type and variants, whereas the detectable anti-HBs HBeAg expression may not occur, but there is maintenance
are targeted against the wild-type viruses. of viral replication, whose existence cannot be measured by
The presence of heterologous subtype-specific antibodies the HBe system [90]. In these cases, HBV/DNA and anti-HBe
could be another factor that could also lead to the co- antibodies can be found in the bloodstream at the same time,
occurrence of HBsAg and anti-HBs in the chronic HBV justifying the occurrence of this atypical serological pattern.
infection context. These antibodies could be directed against The gene ‘C’ of HBV (nucleotide 1814 to nucleotide
HBsAg subtypes different from the coexisting HBsAg, jus- 2450) [82] is divided into the precore region and the core
tifying the serological profile [81, 83, 89]. In these cases, 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 (C1858) stabilizes the ε structure and makes the occurrence
unprocessed core polypeptides (183-aa), which are assem- of a stop codon in the precore a rare event, maintaining the
bled into HBcAg particles, and comprise the virion nucleo- preferred pairing G-C base proposed by Watson-Crick [65,
capsid. Initiation of translation at the first site (nucleotide 94]. However, the substitution of guanine by adenine at
1814) produces a 312 amino acid polypeptide that has a position 1896 (A1896) would result in an unstable base
signal peptide directing it to the endoplasmic reticulum, pairing (A-C), destabilizing the ε structure’s loop and also
where the signal piece is removed by signal peptidases, reducing the efficiency of viral replication [65].
cleaving the N-terminal 19 amino acid residues. With sub- In contrast, the HBV genotypes B, D, and E, and some
sequent processing, the C-terminal 34 amino acid residues strains of genotype C, have a thymidine at nucleotide 1858
are removed, and the resultant polypeptide is secreted as (T1858). Thus, a mutation for adenine at position 1896
HBeAg, a soluble protein, detectable in the bloodstream [85, (A1896) stabilizes the ε structure, forming a new base pair
90]. The function of secretory HBeAg in the viral life cycles (A-T), in parallel, introducing a new stop codon [94] and
is unknown inasmuch as it is not required either for infection increasing the viral replication efficiency [65]. As a result,
or replication. However, it has been suggested that the viral strains which are HBeAg-negative are often found in
HBeAg may have a function in the immune response mod- infections by genotypes B–E. With regard to genotype G,
ulation during chronic HBV infection in adults and in pro- the situation is peculiar, since all isolates are defined by the
moting neonatal tolerance [15]. presence of adenine at nt 1896, which introduces a stop
In the typical course of HBV infection or during antiviral codon natural, making them incapable of expressing HBeAg
therapy in chronic HBV infection, the seroconversion of [94]. However, despite two stop codons (at positions 2 and
HBeAg to anti-HBe is usually accompanied by a decrease 28) in the precore region characteristic of HBV genotype G
in viral replication and hepatic disease remission [90]. [92, 95], the HBeAg can still be detected in the serum of
Among some patients, for unknown reasons, the immune infected individuals by this genotype, but this marker is
pressure associated with seroconversion contributes to the attributed to genotype A, with which the occurrence of co-
viral variants emergence, which are characterized by not infection is frequent [95].
expressing HBeAg or expressing at low levels, although In conclusion, patients infected with mutants in the pre-
these patients have ongoing viral replication associated with core region can then express high levels of DNA/HBV,
liver damage [90, 91]. despite the presence of anti-HBeAg in the serum. However,
During chronic HBV infection, two major types of HBV the cellular immune response modulation, induced by
core gene variants have been frequently reported, which HBeAg against the peptides HBeAg/HBcAg related and
affect the HBeAg expression: the precore mutants and the expressed on the surface of infected hepatocytes, is lost
basal core promoter (BCP) mutants. [96], which can correlate with exacerbation of liver injury
The most prevalent precore region mutation, known as and a worse prognosis [97].
G1896A, is characterized by a guanine-to-adenine transition The second group of mutations affects the BCP (nt 1742
at nucleotide position 1896, which converts codon 28 from to nt 1849), with the double exchange of adenine by thymi-
tryptophan (TGG) to a premature stop codon (TAG), and, by dine at nt 1762 (A1762T) and guanine by adenine at nt 1764
so doing, aborts the translation of HBeAg precursor made of (G1764A) being the most common. These mutations are
29 amino acids coded for by the precore region and the 183 often present in patients with chronic hepatitis or fulminant
amino acids encoded by the core region [92]. The precursor hepatitis, and less frequently in asymptomatic carriers, in
loses amino-terminal 19 amino acids and carboxy-terminal 34 immunosuppressed patients, and in infected individuals de-
amino acids to become HBeAg. This mutation occurs within void of any serological markers [98]. They arise before or
the epsilon (ε) structure, a highly conserved stem-loop at the during the HBeAg seroconversion to anti-HBe and result in
pregenomic RNA, essential for the initiation of encapsidation suppression at the transcriptional level of mRNA coding for
and replication of the HBV pregenome, during the viral rep- the core and precore. The consequence of these changes is
lication cycle [92]. However, the stability of this structure the decrease in HBeAg expression to approximately one-
depends on the strict conservation of base pairing in the stem third of the levels observed in the wild viral strain and, also,
region, since mutations that alter the combination of these contribution to an improvement in the ability of viral ge-
bases may lead to less efficient viral replication or the produc- nome replication [85, 90]. These observations are supported
tion of non-viable viral particles [93]. by transfection studies which show low levels of preC
Thus, in the ε structure, the base guanine, at the nucleo- mRNA as well as of HBeAg secretion in the presence of
tide 1896 (codon 28), on wild-type virus, forms a pair with 1762 T and 1764A [98].
the base thymidine or cytosine of the nucleotide 1858 (co- As previously mentioned, it has been postulated that
don 15), depending on the genotype involved [85]. In gen- HBeAg induces an immune tolerance against itself or to
otypes A, F, and H, the presence of cytosine at nt 1858 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 diminish the function of this transcriptional regulatory ele-
presence in low concentrations in the circulation causes the ment [103], promoting the suppression of viral replication
HBcAg to be targeted directly by the humoral and cellular and the synthesis of HBcAg and HBsAg. Consequently,
immune system, leading to necrosis of hepatocytes and liver patients infected with X-defective HBV mutant are negative
damage [98]. This suggests a close relationship between for HBsAg and anti-HBc, despite the presence of HBV
mutation in the BCP and liver disease advancement, which replication and detectable DNA. X-defective HBV mutants
leads to increased risk of developing hepatocellular carcino- have been isolated from patients with acute or chronic
ma [99]. hepatitis [102].

Profile 6 Serological aspects of clinical and laboratorial


significance
Absence of serologic markers of infection and detectable
HBV/DNA Acute infection and non-detection of HBsAg

HBsAg [−]; HBeAg [−]; anti-HBc [−]; DNA/HBV [+] Classically, 2–3 months after contact with HBV—so still in
the incubation period, even before the biochemical evidence
The isolated detection of HBV/DNA in the absence of of hepatic dysfunction—the detection of HBsAg is possible
serological markers is a typical serological/molecular profile [104]. Usually, it remains detectable in serum until about
and a relatively common laboratory finding, particularly in 2 months after the onset of clinical manifestations. Howev-
the blood transfusion context, when the research of this er, this period may extend to over 3–6 months in individuals
molecular marker is already established in the donors with acute infection [1].
screening. At first, this finding does not seem curious or During this period (i.e., during 2 months after the onset of
unusual, since, in most cases, it can be observed progres- clinical manifestations), the early clearance of HBsAg can
sively the emergence of other serological markers of viral occur before marker identification is made in the laboratory.
replication, which is indicative of infectious blood detection This phenomenon is a result of the inhibition of HBV
still in a recent phase of infection. replication (and decrease of HBsAg), whose mechanisms
However, the situations of persistently detectable DNA, are not well clarified, but are dependent on viral determi-
in the absence of other markers, can define an atypical nants (such as genotype, mutations) [105, 106], and also on
serological profile in HBV infection and lead to the suspi- the host’s immune response, involving the induction of a
cion of mutant virus infection, especially the X-defective CD8(+) T-cell response (key cellular effectors mediating
HBV mutant, whose characteristic is briefly described HBV clearance from the liver) by CD4(+) cells (the master
below. regulators of the adaptive immune response to HBV), as was
The X-ORF (X-region) encodes for a 154aa protein demonstrated by Yang et al. [107]. Therefore, the negativity
called HBx (protein X). Protein X is known to transactivate of this marker does not exclude the diagnosis of acute HBV
transcriptional regulatory elements, which include the en- infection!
hancer and core promoter elements. The core promoter,
located at nt 1586 to nt 1849 [98, 100], regulates the tran- Detection of IgM anti-HBc antibodies: acute or chronic
scription of the core/pregenome, and enhancer II, located at infection?
nt 1687 to nt 1805 [101], is implicated in the activation of
the S-promoter [98, 101]. The sequence nt 1742 to nt 1849 As already mentioned, IgM antibodies appear early, even
is called the BCP. It is supposed to be sufficient for the during the incubation period. When the disease is clinically
correct transcription initiation of the pregenome and precore expressed, IgM and IgG antibodies are usually present.
mRNA [82]. DR1 (direct repeated) and DR2, which are After 2–6 months, IgM disappears, leaving only IgG anti-
implicated in the origin of HBV DNA, are also located in bodies [4].
the X-region. In contrast to most viral infections, patients with acute
The X-defective HBV mutant is commonly characterized and chronic HBV often produce both IgM class and IgG
by an 8-nt deletion between nt 1770 and 1777 and a point class anti-HBc antibodies; therefore, the mere presence of
mutation of DR2 (T-to-C) in the X-ORF [102]. This deletion IgM anti-HBc is not diagnostic of an acute infection. How-
results in a C-terminally truncated X protein of 134aa (due a ever, higher levels of IgM anti-HBc are generally produced
loss of 23aa and the addition of three normal aa), which during the acute phase as compared with chronic infection,
causes a loss of its transactivating activity. The 8-nt deletion and this quantitative difference can be the only serological
of the core promoter/enhancer II complex sequence may means of differentiating an acute HBV infection from an
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Eur J Clin Microbiol Infect Dis

acute exacerbation of a chronic infection. Thus, the positiv- not develop in all HBV-infected patients, or may appear at
ity for IgM anti-HBc antibodies should be analyzed with various times after the appearance of anti-HBc. The serocon-
caution so as not to induce errors in diagnosis. On the other version to anti-HBs antibodies indicates viral elimination.
hand, in practice, anti-HBc IgM negativity is important, However, these antibodies can become undetectable in
because if this indicator is negative, the probability of acute patients who have recovered fully from infection. Although
infection in HBsAg-positive cases is zero [104]. there is still limited knowledge about the kinetics and regula-
tion of HBs/HBe-specific antibody response [109], the low
Correlation between anti-HBc and anti-HBe antibodies HBs and HBe immunogenicity [110, 111] comprises one of
production several potential immunological mechanisms that could ex-
plain the non-development or non-detection of these antibod-
Although HBcAg and HBeAg are serologically distinct, ies in the clinical course of HBV infection. Regardless of this
they are structurally related, since these two proteins keep factor, it has been shown that antibody production to viral
in its structure a very extensive homology in the amino acid protein HBe and HBs is a T-cell-dependent process [28], and
residues sequence [53]. This has been observed from experi- that the Th-cell response to HBcAg/HBeAg supports the
ments that demonstrate the existence of cross-reactivity in production of HBc and HBe antibodies, as well as anti-
recognition of these antigens at the Th-cell level for anti- envelope antibodies [112]. Considering that HBc/HBeAg-
bodies production [28, 53], although the immune responses specific Th cells in humans mediate anti-envelope antibodies
to these antigens appear to be regulated independently. production, and that there is a significant correlation between
It is known that anti-HBc antibody can be produced via the activation of CD4+ T lymphocytes by HBcAg/HBeAg
T-cell-independent and T-cell-dependent pathways, whereas and the seroconversion phase to anti-HBe and anti-HBs in
antibody production to the HBeAg is strictly T-cell- acutely infected patients [11], it is tempting to postulate that
dependent [28]. Thus, the cross-reactivity of Th-cell recog- the seroconversion to anti-HBe antibodies predicts the sero-
nition of HBcAg and HBeAg predicts a correlation between conversion to anti-HBs antibodies. Nevertheless, it is possible
the T-cell-dependent pathway of anti-HBc antibody produc- that anti-HBe antibodies can be detected even in the absence
tion and anti-HBe antibody production. Accordingly, the of anti-HBs antibodies [113, 114], or that anti-HBs antibodies
emergence of anti-HBe antibodies is associated with an are present alone in the serological profile (or in combination
increasing T-cell response to the core protein, and is accom- with other markers, other than anti-HBe), without which these
panied by a slight increase in anti-HBc antibodies [11]. serological situations are necessarily characterized as atypical
Therefore, low anti-HBc titers may be consistent with only [114].
T-cell-independent anti-HBc production, may reflect a def-
icit in HBe/HBcAg-specific Th-cell function, and are usu-
ally related to anti-HBe negativity in the serological profile. Prevalence of atypical serological profiles in HBV
On the other hand, high titers of anti-HBc antibodies are infection
associated with its production via T-cell-independent and T-
cell-dependent pathways, and correlates with the presence Atypical serological profiles in HBV infection include sero-
of anti-HBe antibodies in the serological profile [23], sup- logical situations that tend to occur most frequently in the
porting the theory that, if T-cells assist in the anti-HBc chronic infection context in response to a long history of
production, they would presumably also assist in the anti- HBV infection, associated with virological characteristics of
HBe production (Fig. 3). These data demonstrate that, al- the agent (involving persistent viral replication, replication
though the anti-HBe antibodies presence is observed in only mechanism, natural variability, and wide capacity of muta-
33 % of anti-HBc-positive individuals [108], its detection tion), but also due to an inadequate HBV-specific immune
presupposes the existence of cellular immune mechanisms pressure of the host. And that is what the serological find-
responsible for HBV clearance, suggests convalescence, and ings observed in studies conducted in regions of high prev-
progresses toward recovery, whereas its absence in the pro- alence of HBV infection (where the majority of infections
file may suggest the opposite. Obviously, the anti-HBe are contracted perinatally or in early childhood) have sug-
detection in the absence of anti-HBc antibodies suggests a gested, demonstrating that, in chronic carriers, involving
profile initially incoherent and likely to be revised. patients with chronic active hepatitis, or with chronic per-
sistent hepatitis or asymptomatic carriers, the presence of
Seroconversion to anti-HBe and anti-HBs antibodies atypical patterns is more incident [86, 115, 116]. However,
these serological patterns can be observed in any clinical
The seroconversion to anti-HBe antibodies are frequently setting of HBV infection, since its occurrence may also be
correlated with viral clearance and remission of liver disease associated with the agent [117] and/or to the host [118], but
during chronic infection. However, antibodies to HBeAg may not just due to the long history of HBV infection.
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Eur J Clin Microbiol Infect Dis

structural relationship

HBc antigen HBe antigen


2 1
4

T-helper-cells B-cells T-helper-cells [CD4+]


HBe/HBcAg specific
HBe/HBcAg
IgM anti-HBc antibodies 5
specific

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 production via T-cell-independent as well as T-cell-dependent path-
antibodies via T-cell-dependent pathways. 1 HBcAg directly stimulates ways (high immunogenicity); 4 HBeAg requires T-cell helper function
B cells to produce anti-HBc antibodies (primarily IgM); 2 HBcAg in the anti-HBe antibodies production; 5 emergence of anti-HBe anti-
requires T-cell helper function (which may be variably present from bodies (in correlation with high titers of anti-HBc) supported by the
patient to patient) in the production of IgG anti-HBc antibodies; 3 cross-reactivity in recognition of these antigens at the T-helper-cell
demonstrating its property to stimulate the anti-HBc antibodies level

Despite these conjectures, there are no data on the prev- deletions when compared with other genotypes [124].
alence of these patterns (or which patterns are more preva- Since deletions in preS/S has been a factor to justify the
lent) in the context of acute or chronic HBV infection, since co-occurrence of HBsAg and anti-HBs in the bloodstream
its occurrence, in most cases, have only been mentioned in [79, 84], it seems plausible to associate a greater possibility
the context of other investigations on HBV [116, 119–121] of detection of atypical serological patterns in the occurrence
or identified primarily by routine laboratory tests in the of infection with this genotype [30].
context of HBV diagnosis [122] (or accidentally, as a result However, it is observed that there is a poor relationship
of screening protocols [123]), but not from serological stud- between atypical patterns and genotypes of HBV, and, even
ies correlating the incidence of atypical serological profiles if there was such an association, the investigation of the
in a population of HBV-infected individuals. 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.
Atypical serological profile and HBV genotypes Even so, this correlation deserves further investigation.

So far, there has been no study carried out to establish any


relationship between the presence of atypical patterns and Conclusion
genotype of the infecting HBV. However, it seems logical to
assume that infection by certain genotypes, due to some The natural course of hepatitis B virus (HBV) infection is
specific peculiarities, may justify this finding and even favor directly linked to immune tolerance, infection by viral var-
its appearance. For example, with regard to infection by the iants, and viral clearance. The Immune tolerance, infection
G genotype (possibly also by B and E), the occurrence/ by viral variants, and viral clearance, in turn, comprise
presence of mutations in the precore region (or BCP) is factors which are closely related to DNA levels produced
expected and may comprise a frequent finding in some by the agent, genetic expression of viral antigens, and also
populations, especially in France and the USA, where in- the patient’s immune response. The interrelationship of
fection by that genotype is incident [120]. Therefore, the these factors, most of the time, may justify the identification
association of infection by this genotype with the coexis- of atypical serological patterns which are beyond those
tence of anti-HBe and DNA in the circulation, may be wide. classically known and universally accepted, such as those
Another illustrative example is related to HBV/C genotype described here. Thus, it seems possible that acute or chronic
infection. HBV/C has been given some peculiarities, among carriers of HBV are identified, showing serological patterns
which are the highest frequency of BCP mutations and preS/S that do not characterize them as such.
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Eur J Clin Microbiol Infect Dis

Moreover, the advent of the next generation of immuno- 14. Ni YH, Hsu HY, Chang MH, Chen DS, Lee CY (1993) Absence
or delayed appearance of hepatitis B core antibody in chronic
assays, the improvement in the sensitivity and specificity of
hepatitis B surface antigen carrier children. J Hepatology 17
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HBV also tend to reveal other curious serological situations, 15. Milich DR, Chen MK, Hughes JL, Jones JE (1998) The secreted
such as HBsAg detection in the absence of HBV/DNA hepatitis B precore antigen can modulate the immune response to
the nucleocapsid: a mechanism for persistence. J Immunol
[125], and which will be added to a constellation of atypical
160:2013–2021
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17. Coursaget P, Yvonnet B, Bourdil C, Mevelec MN, Adamowicz P,
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18. Echevarría JM, León P, Domingo CJ, López JA, Echevarría JE,
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Conflict of interest The author has declared that they have no infections in Spain. J Med Virol 33(4):240–247
conflict of interest. 19. Fiordalisi G, Primi D, Tanzi E, Magni E, Incarbone C, Zanetti
AR, Cariani E (1994) Hepatitis B virus C gene heterogeneity in a
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