Peculiarities in the designations of hepatitis B virus genes, their products, and their antigenic specificities: a potential source of misunderstandings.

The nomenclature of the hepatitis B virus (HBV) genes and their products has developed stepwise, occasionally in an erratic way, creating many misunderstandings, especially among those who do not know the structure of HBV and its genome in detail. One of the most frequent misunderstandings, even presented in leading journals, is the designation of HBV "e"-antigen as envelope or early antigen. Another problem area are the so-called "pre" regions in the HBV genome present upstream of both the core and the surface genes of HBV, inadvertently suggesting that they may be a part of corresponding precursor proteins. Misnomers and misclassifications are frequent in defining the subgenotypes and serological subtypes of HBV. Even the well-established terminology for HBV surface (HBs) or HBV core (HBc) antigen deviates from the conventional virological nomenclature for viral envelopes or capsid proteins/antigens, respectively. Another matter of undesirable variability between publications is the numbering of the nucleotides and the graphical representation of genomic maps. This editorial briefly explains how the nomenclature evolved, what it really means, and suggests how it could be adapted to today's knowledge.

Actualización sobre hepatitis B / Upgrade in hepatitis B

El virus de la hepatitis B (VHB), es un virus DNA, el cual tiene varios antígenos, como el antígeno de superficie, y antígeno core. Colombia, es un país de baja endemicidad, sin embargo, en la Sierra Nevada de Santa Marta, está endemicidad es alta. El VHB tiene como una de sus complicaciones la hipertensión porta. En general, el VHB no atraviesa la placenta, por lo que la infección es rara in utero. Son pocos los pacientes que se presentan con HB y falla hepática fulminante y por lo tanto, son pocos los antivirales que han sido utilizados, con muy poca experiencia.

The hepatitis B virus (HBV) is a DNA virus, which has several antigens such as surface antigen and core antigen. Colombia is a country of low endemicity, however, in the Sierra Nevada of Santa Marta, is endemic is high. HBVis one of the complications of portal hypertension. In general, HBV does not cross the placenta, so the infection is rare in utero. Few patients who present with HB and fulminant hepatic failure and therefore, few antiviral drugs that have been u s e d , wi t h v e r y l i t t l e e x p e r i e n c e.

Utilizing self-prepared ELISA plates for a cross-population study of different anti-HBe IgG subclass profiles.

Fourteen serum samples obtained from hepatitis B virus (HBV) chronic carriers and patients recovered from hepatitis B infection were used with four sodium dodecyl sulfate-treated enzyme-linked immunosorbent assay (ELISA) plates available commercially, and one self-prepared HBcAg analog for evaluation of anti-HBe subclass pattern absorbance. The self-prepared plates had the best performance and were thus used for samples obtained from 104 (60 male and 44 female) HBV chronic carriers and 439 (247 male and 192 female) recovered individuals. Tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also carried out in 21 of the subjects (>25 IU/ml). Statistical comparison of these patients with elevated ALT/AST levels with other ALT/AST-normal chronic carriers revealed no significant differences in the anti-HBe OD, although the mean optical density (OD) of patients with elevated ALT/AST levels was higher. The results suggest that the anti-HBe IgG subclass profiles in the chronic carriers did not change with inflammation of the liver, and were independent of sex and age. In contrast to previous anti-HBc findings, the distribution pattern of anti-HBe subclasses in HBV chronic carriers was IgG1 > IgG4 > IgG3 while in the recovered individuals it was IgG1 > IgG3 > IgG4, for both males and females. Subclasses IgG1 and IgG2 were the most and least prevalent isotypes, respectively, in both study groups. The results of the study suggest that induction of IgG1 and/or IgG3 antibodies is important for effective virus neutralization, while IgG2 antibodies are of limited importance. Significantly higher OD values for anti-HBe IgG4 were observed when comparing samples from the chronic carriers and recovered individuals, which may reflect the effects of persistence. Further, in contrast to previous anti-HBs results, the concentrations of total IgG and IgG1 were higher in the samples from chronic carriers relative to those from recovered individuals.

Study of IgG subclass profiles of anti-HBs in populations with different HBV infection status.

To study IgG-specific subclasses of hepatitis B virus (HBV) surface antigen (anti-HBs), in different populations in Taiwan, a comparison was made between 104 chronic carriers (60 male and 44 female) and 439 recovered individuals (247 male and 192 female). Biochemical analyses of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also performed. Among the 104 chronic carriers, 21 patients had abnormal ALT and AST levels (> 25 IU/ml). When comparing the patients with abnormal ALT and AST levels to chronic carriers with normal ALT and AST levels, no statistical difference was observed for anti-HBs levels (p > 0.05). The IgG subclass pattern of the relative anti-HBs IgG subclass titers was IgG1 > IgG3 = IgG4 in both chronic carriers and recovered individuals (p < 0.05). IgG1 is the predominant anti-HBs antibody after HBV infection, in either chronic carriers or in HBV-cured individuals. This finding is partly inconsistent with data reported from other group who suggested in individuals naturally infected, the anti-HBs IgG consists mainly of IgG3 and IgG1. In contrast to that of our previous studies of anti-HBe and anti-HBc, the mean OD values of anti-HBs total IgG, and all IgG subclasses except for IgG2, of either males or females, were significantly higher in recovered individuals than in chronic carriers, while the mean OD values of anti-HBe and anti-HBc were significantly higher in chronic carriers than in recovered individuals (P < 0.05). The IgG subclass profile of anti-HBs in chronic carriers was not changed with liver inflammation and was independent of sex and age, except in individuals with abnormal ALT and AST for whom anti-HBs IgG1 was not significantly higher than IgG3 (p > 0.05), in spite of that whose mean O.D. value is higher.

The IgG subclass profile of anti-HBs response in vaccinated children and children seroconverted after natural infection.

The IgG subclass profiles of anti-HBs antibodies were investigated in 30 children who had recovered from acute hepatitis B and 40 children vaccinated against hepatitis B virus (HBV) with Engerix B. After natural seroconversion the mean geometric value of anti-HBs titres was ca 41-fold lower than at the peak of response in vaccinees, and specific antibodies were highly restricted to IgG1 subclass followed by IgG3 with only a minor contribution of IgG2 and IgG4. Conversely, in children immunized with recombinant HBsAg, IgG1 and IgG3 dominated after two doses of vaccine and 1 month after the third injection but the response was less selective and more variable. One year after vaccination IgG4 anti-HBs antibodies became the second dominating isotype. Significant statistical differences in the profiles of IgG anti-HBs were observed when the age and maturity of humoral response were considered. While children vaccinated below 5 years of age responded mainly with IgG1 and IgG3 subclasses, older children (> 5 years) showed a high individual variability in the specific profiles with a high contribution of IgG4. We concluded that vaccination at a younger age leads to the production of antibody subclasses which are more effective for virus neutralization.

The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype.

Secretion of the hepatitis B virus (HBV) e antigen (HBeAg) has been conserved throughout the evolution of hepadnaviruses. However, the function of this secreted form of the viral nucleoprotein remains enigmatic. It has been suggested that HBeAg functions as an immunomodulator. We therefore examined the possibility that the two structural forms of the viral nucleoprotein, the particulate HBV core (HBcAg) and the nonparticulate HBeAg, may preferentially elicit different T helper (Th) cell subsets. For this purpose, mice were immunized with recombinant HBcAg and HBeAg in the presence and absence of adjuvants, and the immunoglobulin G (IgG) isotype profiles of anti-HBc and anti-HBe antibodies were determined. Second, in vitro cytokine production by HBcAg- and HBeAg-primed Th cells was measured. The immunogenicity of HBcAg, in contrast to that of HBeAg, did not require the use of adjuvants. Furthermore, HBcAg elicited primarily IgG2a and IgG2b anti-HBc antibodies, with a low level of IgG3, and no IgG1 anti-HBc antibodies. In contrast, the anti-HBe antibody response was dominated by the IgG1 isotype; low levels of IgG2a or IgG2b anti-HBe antibodies and no IgG3 anti-HBe antibodies were produced. Cytokine production by HBcAg- and HBeAg-primed Th cells was consistent with the IgG isotype profiles. HBcAg-primed Th cells efficiently produced interleukin-2 (IL-2) and gamma interferon (IFN-gamma) and low levels of IL-4. Conversely, efficient IL-4 production and lesser amounts of IFN-gamma were elicited by HBeAg immunization. The results indicate that HBcAg preferentially, but not exclusively, elicits Th1-like cells and that HBeAg preferentially, but not exclusively, elicits Th0 or Th2-like cells. Because HBcAg and the HBeAg are cross-reactive in terms of Th cell recognition, these findings demonstrate that Th cells with the same specificity can develop into different Th subsets based on the structural form of the immunogen. These results may have relevance to chronic HBV infection. Circulating HBeAg may downregulate antiviral clearance mechanisms by virtue of eliciting anti-inflammatory Th2-like cytokine production. Last, the influence of antigen structure on Th cell phenotype was not absolute and could be modulated by in vivo cytokine treatment. For example, IFN-alpha treatment inhibited HBeAg-specific Th2-mediated antibody production and altered the IgG anti-HBe isotype profile toward the Th1 phenotype.

Subclasses of antibodies to hepatitis B core antigen in chronic HBV infection: changes during treatment with alpha interferons and predictors of response.

Response to interferon therapy in chronic hepatitis B virus (HBV) carriers is preceded by the appearance of IgM class anti-HBc (antibody to hepatitis B core antigen). The temporal relationship and magnitude of the IgM anti-HBc response is variable suggesting that the antibody is not directly involved in hepatocyte lysis, but is merely a marker of a changed state of immunity to the nucleocapsid proteins, induced by interferon. IgG 1, 2, 3, and 4 class anti-HBc did not change during therapy, but IgG 3 anti-HBc was significantly lower in responders than non-responders. IgG anti-HBc of all subclasses was absent in two Chinese HBV carriers. Lower than normal titres of anti-HBc (p less than 0.001) were detected in human immunodeficiency virus antibody positive (anti-HIV) HBV carriers. These data indicate the presence of altered immunity to the nucleocapsid antigens in these two types of chronic HBV carrier that are known to respond poorly to antiviral therapy.

Significance of various classes of anti-HBc in individuals with chronic asymptomatic HBV infection.

Among 108 individuals with chronic asymptomatic hepatitis B virus infection, liver biopsies and serological investigation were performed simultaneously to determine the correlations of anti-HBc with the infection level of the liver pathology. The results showed that the prevalence of IgA and IgM anti-HBc, and the titers of IgA, IgM, and total anti-HBc, correlated with the liver damage. IgA anti-HBc was most prominent, being present in the whole group of patients with chronic active hepatitis with cirrhosis, and was absent in the whole group of asymptomatic carriers with normal histology. There were no linkages of IgA and IgM anti-HBc with the infectivity; in addition, the titer of total anti-HBc correlated inversely with the viral replication.

IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections.

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses.

A human monoclonal anti-HBsAg antibody (IgG1, lambda) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgG1 antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.

Proportions of IgG subclasses in antibodies formed during vaccination with hepatitis B surface antigen.

Nineteen healthy adults were given three injections of hepatitis B vaccine (days 0, 30, and 180), and serum samples were obtained on days 0, 21, 51, and 201. A fourfold or greater increase of IgG antibodies within 201 days was detected in 18 individuals (geometrical mean increase 26-fold), but final IgG antibody concentrations (0.3-3 micrograms/ml) were low. Some vaccines had almost reached peak concentration on day 21, in others a great increase was observed between days 21 and 51, and in a few individuals between days 51 and 201. IgG1 was the predominant subclass in the antibodies of most individuals. Ninety-five per cent of all IgG antibodies were IgG1 in early samples of early responders. In late samples of all vaccinees the average proportion of IgG1 antibodies was 78%. The proportion of IgG2 antibodies was slightly higher in late samples (average 12%) than in the early samples of the early responders, (average less than 6%, P less than 0.05). Considerable amounts of IgG3 and IgG4 antibodies were found in one vaccine, but on the average the proportion of IgG3 or IgG4 antibodies was low (less than 6%).

Quantitation of antibody isotypes in solid-phase assays. Comparison of myeloma protein and monoisotypic antibody standards.

Two methods have been proposed for the standardization of isotype specific antibody assays. In one, myeloma proteins directly attached to plastic surfaces are used as standards, whereas the other method employs antigen coated surfaces followed by monoisotypic antibodies as standards. These standardization methodologies have been investigated by submitting 4 monoisotypic human antibodies to a solid-phase assay standardized by the myeloma method. Specific antibody concentrations of each were determined so that each could serve as a monoisotypic standard. Three purified monoclonal mouse antibodies were also tested which allowed use of the same preparation as a monoisotypic antibody standard or as a 'myeloma protein' standard. Ten times more myeloma protein than specific antibody is needed for the same level of binding of the anti-isotype antibody. Therefore, assays standardized with myeloma proteins give erroneously high concentrations for sample antibodies. The same concentration of antibodies of different specificities (used with different antigen coats) gave very comparable levels of binding of the labeled antibody. This supports the claim that for quantitation of antibodies an antibody standard can be used that is of different specificity to the sample antibody to be measured.

Immunoglobulin G subclass restriction of antibodies against hepatitis B surface antigen.

Immunoglobulin G (IgG) antibodies against hepatitis B surface antigen were found to be restricted to subclasses IgG1 and IgG3 in serum samples of 17 individuals. Quantitative determinations showed that in 10 samples the minor serum subclass IgG3 contained more antibodies than the predominant subclass IgG1. Ranges of concentrations were between 0.72 and 16.50 micrograms/ml for IgG3 antibodies and between 0.55 and 6.19 micrograms/ml for antibodies of subclass IgG1.

Monoclonal antibodies to hepatitis B surface antigen (HBsAg) as a tool for the automated HBsAg screening.

Hybridomas secreting monoclonal antibodies to hepatitis B surface antigen (HBsAg) were established by fusion of the spleen cells from mice immunized with purified HBsAg with the mouse myeloma cell line P3-NSI/1-Ag4-1. The monoclonal antibodies to the group-specific antigen (a) produced by one of them were used for the automated screening of HBsAg on the Groupamatic 360 (Kontron International). Its sensitivity is almost equal, but slightly inferior, to the system employing polyclonal horse antibodies to HBsAg; it barely detects 6 ng/ml of HBsAg. It is also as highly specific as the system with polyclonal antibodies; the incidence of false-positive reactions is 0.2%. These results indicate that the monoclonal antibodies will become a practical source for the HBsAg screening on the Groupamatic.

Production of monoclonal antibodies against hepatitis B surface antigen (HBsAg) by somatic cell hybrids.

Hybridomas secreting anti-HBs were produced by fusion of either adw or ayw HBsAg primed mouse spleen cells with either P3 X63 Ag8 or P3 NSI 1 Ag4 1 mouse myeloma cell lines. Individual anti-HBs secreting clones were isolated by limiting dilution procedures, and six cell lines have been established, namely, BX182, BX259, BX248, CN324, DN283, and DN296. Progenies of each cell line were derived from a single clone obtained from three subclonings of six anti-HBs positive initial fusion colonies. Clones were passaged in tissue culture and as tumors in syngeneic mice for upwards of six months. Anti-HBs of each line showed characteristic reactivity (detection) patterns in radioimmunoassay using different antigen subtype solid phases followed by either 125I-HBsAg or 125I-goat anti-mouse IgG probe. The specificity of the anti-HBs from each clone for the subdeterminants of HBsAg was identified by their reaction with 125I-HBsAg ligands of several subtypes in a radioimmunoprecipitation assay. Four types of reaction were identified and correlated to the conventional serological subtyping definitions; they were anti-HBs/a (BX259 and CN324), anti-HBs/d (BX182), and possibly anti-HBs/w (BX248 and DN296) and anti-HBs/y (DN283). These monoclonal antibodies will be important for the elucidation of the antigenic structure of native HBsAg and will provide valuable reagents for both antigen detection and subtyping.

Epidemiological survey of hepatitis B virus infections in various groups of population.

An epidemiological survey of hepatitis B virus (HBV) infection has been carried out from 1974 up to 1978 in four laboratories, with the aim of identifying high-risk groups in the population. Radioimmunoassay procedures were used in order to detect the hepatitis B surface antigen (HBsAg) and the corresponding antibody (anti-HBs) in serum samples from over 100,000 individuals of various geographical origin (mainly from Ligury or from other areas in Northern Italy). The groups of population under monitoring included: individuals without hepatic illness just after admittance into hospitals (these groups were found to be adequately representative of the corresponding open population), groups of children and boys from the open school population, individuals living in various communities (foundling hospital, children college, recruits, institutionalized old people, subnormal individuals and their assistance staff), non-assistance working categories (workers from metallurgical and chemical industries, shipped seamen), hospital assistance personnel, dialyzed and transplanted renal patients, blood donors. The results obtained showed a considerable variability of HBV infections among the examined groups of population. In particular, the frequency of HBsAg and/or anti-HBs detection appeared to be significantly affected by each of the following epidemiological parameters: geographical area, sex, age, pregnancy, life in communities and contacts with hepatitis patients, mental deficiency, non-assistance working activities, assistance activity in the hospital environment, hemodialysis and transplants, selection of population groups by HBsAg screening.

Hepatitis B-antigen subtypes in Australia as detected by radioimmunoassay.

The distribution of the major subtypes of hepatitis B surface antigen (HBsAg) was examined in asymptomatic white Australian blood donors and patients with liver disease to attempt ot assess the biological significance of these subtypes in this relatively homogeneous Caucasian population. Since previous studies have shown a very low prevalence of HBsAg in white Australians, a highly sensitive solid phase radioimmunoassay (RIA) was developed and used to subtype the antigen. A total of 46 samples of hepatitis B-antigen positive sera, obtained from 21 asymptomatic volunteer blood donors and 25 patients with liver disease, were tested. Sixteen of these 21 asymptomatic carriers were shown to have subtype "ad" whereas only seven of the patients with liver disease had this subtype. This difference was significant (P less than 0.005) and supports the view that subtype "ad" is associated with a carrier state and subtype "ay" with liver disease in Australia.