HLA antigens and the antibody response after vaccination to hepatitis B

The aim of our work was the search for immunogenetic factors that influence the antibody response to HBs antigen. We analyzed the HLA-A, -B, and -DR antigen frequencies in 19 seropositive to HBs and 28 seronegative to HBs healthy persons finding an elevated frequency of B5 in the seropositive group (p value 0.037). After vaccination with Hevac B Pasteur vaccine of the seronegative persons, the low antibody response was associated with B13 (p value 0.041). An association between local side reactions to the vaccine and HLA A3 and B35 were also found (p values 0.042 and 0.022 respectively). The presented p values are not significant after correction for the number of antigens tested and for this reason our findings require confirmation in an independent study.     

Dissociated antibody responses to the S and pre-S2 regions of the hepatitis B virus after vaccination in hemophiliacs

The membranes of hepatocytes and the pre-S2 envelope protein of the hepatitis B virus (HBV) contain binding sites for polymerized human albumin, which is thought to act as a link between HBV and hepatocytes. Hence, anti-pre-S2 antibodies should prevent HBV uptake by the liver, and there is indeed preliminary evidence that they protect chimpanzees from HBV infection. To evaluate whether a plasma-derived vaccine containing the pre-S2 sequence induced an anti-pre-S2 response in 105 vaccinated hemophiliacs, anti-pre-S2 was measured in parallel with antibody to hepatitis B surface antigen (anti-HBs). Eighty-five percent of the hemophiliacs had both anti-pre-S2 and anti-HBs when vaccination was completed, 13% had anti-HBs alone, and 2% (two cases) had anti-pre-S2 alone. Eighty-seven percent of anti-pre-S2-positive hemophiliacs compared with only 50% of anti-pre-S2-negative hemophiliacs (P less than 0.001) developed high anti-HBs titers (greater than or equal to 1,000 mlU/ml). This study demonstrates, therefore, that the antibody responses to the S and pre-S2 regions of HBV may be dissociated after vaccination in hemophiliacs and that higher anti-HBs titers are attained in anti-pre-S2-positive hemophiliacs.     

Hepatitis B vaccination of immunosuppressed heart transplant recipients with the vaccine Hepa Gene 3 containing pre-S1, pre-S2, and S gene products

The antibody response of immunosuppressed heart transplant recipients to vaccination with the hepatitis B (HB) virus vaccine Hepa Gene 3 (HG-3), containing HB virus pre-S1, pre-S2, and S gene products, was examined. Three heart transplant recipients who had been vaccinated preoperatively against HB responded well to the vaccination. Five of 38 patients (13.2%) vaccinated postoperatively before HG-3 vaccination with the second-generation vaccine Gen-H-B-Vax-D (37 without and 1 with detectable anti-HBs response) and 3 of 24 (12.5%) without previous HB vaccination developed protective anti-HBs titers (greater than 10 U/1) after immunization with the HG-3 vaccine. The l low response rate (8/62, 12.9%) found for postoperatively vaccinated patients indicates that heart transplant recipients should be vaccinated against HB before immunosuppressive medication.Abbreviations HB hepatitis B - HG-3 Hepa gene 3     

In vitro immune responses to hepatitis B surface antigen (pre-S2 and S) following remote infection by hepatitis B virus in humans

In this report we evaluate the human immune response to hepatitis B surface antigen (HBsAg) following remote infection with hepatitis B virus (HBV). HBsAg-reactive lymphocytes can be readily demonstrated in the peripheral blood of individuals with established immunity following infection with HBV.In vitro stimulation with small doses of plasma-derived HBsAg, yeast-derived HBsAg (S region) or pre-S2 peptide will induce specific IgG to HBsAg (anti-HBs) in the absence of a polyclonal increase in total IgG. The pre-S2 peptide will stimulate, in a T cell-dependent fashion, thein vitro production of anti-HBs with specificity for the S domain. This anti-HBs production is mediated by pre-S2-stimulated soluble T-cell factors. Peripheral blood mononuclear cells from individuals with established immunity proliferate to the yeast-derived HBsAg but not to the plasma-derived HBsAg or pre-S2 peptide. The chronic HBsAg carriers do not produce anti-HBs following stimulation with HBsAg regardless of the source or component of antigen used. Different study protocols failed to demonstrate HBsAg-specific responses in the peripheral blood mononuclear cells of chronic carriers.     

Long-term immune reactivity to pre-S(2)-antigen after acute hepatitis B infection

We investigated sera from 39 patients, taken 1-8 years after recovery from acute hepatitis B for anti-pre-S(2) by Western blotting and for anti-HBs by radioimmunoassay. Anti-pre-S(2) antibodies were found in 27 out of 39 sera (69%) with the highest frequency in sera with anti-HBs greater than 100 IU/I (92%). However, sometimes sera with low anti-HBs titres showed a strong response in Western blotting. Acute hepatitis sera were also investigated from a limited number of patients (n = 14). Anti-pre-S(2) antibodies were found during antigenaemia (four out of six patients) and within 3 months after the maximum of alanine amino transferase (ALAT) (seven out of ten patients). Anti-pre-S(2) is an early antibody. It remains in the circulation for many years similar to but independent of anti-HBs.     

HLA and the rubella-specific immune response following vaccination

A group of 88 seronegative 11-year-old Dutch girls was selected to be tested for human leukocyte antigens (HLA) and for both the in vivo and in vitro immune response to rubella virus, 6 and 12 weeks after rubella vaccination. Although a slight influence of HLA-associated factors on the antibody levels, as measured by the enzyme-linked immunosorbent assay could not be excluded, no evidence for HLA-associated control of the rubella-specific immune response following vaccination was obtained. From these data it is concluded that HLA-associated factors cannot be expected to hamper the effectiveness of large-scale rubella vaccination procedures.     

Hepatitis B pre-S1 and pre-S2 proteins: clinical significance and relation to hepatitis B virus DNA

Pre-S proteins may have an important role in virus assembly and virus entry into the host cell. The presence of pre-S proteins in serum has also been thought to correlate with active viral replication. To investigate whether pre-S proteins in serum might have additional diagnostic and/or predictive value for liver sequelae in HBV infection, sera from six different serological groups of patients with HBV markers (total number 363) and different manifestations of liver histology were examined for the presence of pre-S1 and pre-S2 proteins using micro-ELISAs. Pre-S1 and pre-S2 proteins were detected significantly more often in HBV-DNA-positive than in HBV-DNA-negative sera from HBsAg carriers. However, pre-S1 and pre-S2 proteins were also found in HBV-DNA-negative HBsAg carriers irrespective of serum HBeAg/anti-HBe or liver histologic findings. These results suggest that the presence of the pre-S1 and or pre-S2 proteins in serum either does not seem to reflect the presence of active viral replication and active liver disease or pre-S proteins are more readily detectable than HBeAg and HB-DNA as measured by a dot-blot technique. Furthermore, the presence of pre-S proteins in serum is strongly correlated with that of HBsAg.     

An immune response gene linked to MHC in man

With a view to finding a relationship between immune response and MHC in man, 83 D negative mothers with allo-into-D antibodies and 26 PLA1 negative mothers with allo-anti-PLA1 antibodies were investigated as regards their HLA-A, B and DR antigens. We have found that there is a highly significant relation between the DR3 antigen and an immune response to the PLA1 antigen, but none to the D antigen.     

Translation products of pre-S(1), pre-S(2) regions and the S gene of hepatitis B virus: susceptibility of their antigenic activities to treatment with heat, urea formalin or pepsin

Hepatitis B subviral particles, purified from plasma of asymptomatic carriers seropositive for hepatitis B e antigen, were treated with various conditions reported for the processing of vaccines. Thereafter, antigenic activities displayed by the translation products of pre-S(1), pre-(2) regions and the S gene were determined with monoclonal antibodies, and the reactivity for polyalbumin receptor was tested. Heating at 100 degrees C for 1.5 min and then at 65 degrees C for 10 h preserved more than 1/2 of antigenic activities representing products of pre-S(1), pre-S(2) regions and the S gene. After incubation in the presence of 8 M urea at 37 degrees C for 4 h, more than 2/3 of antigenic activities still remained. The antigenic activity of the S gene product was decreased to 2/3 and that of pre-S(2) region product to 1/3, after treatment with formalin at the final concentration of 1:4000 at 37 degrees C for 72 h, whereas the activity of pre-S(1) region product was affected drastically. Although 1/5 of the antigenic activity of the S gene product survived the digestion with pepsin for 18 h, antigenic activities of pre-S(1) and pre-S(2) region products were destroyed almost completely. Polyalbumin receptor, borne by the pre-S(2) region product, was lost by pepsin digestion also. Based on the results obtained, heating may be most appropriate for sterilizing plasma-derived hepatitis B particles for use as a vaccine, because it is reliably virucidal and would not affect the protective efficacy to an extent as the other virucidal methods would.     

Change in immune response to hepatitis B in boys with haemophilia

Eleven of 27 haemophilic boys who received a common batch of Factor VIII concentrate subsequently developed acute hepatitis B; although 9 were considered not to have been previously exposed to the virus, 2 other boys had been considered immune to hepatitis B. The amount of concentrate received by each child, together with their HIV-antibody status and T-lymphocyte subset distribution prior to exposure, did not influence their response to the hepatitis B virus (HBV). The two previously immune children who became infected, however, had evidence of the HIV-associated persistent generalized lymphadenopathy syndrome. Detailed investigation of the suspect batch of concentrate revealed hepatitis B surface antibody to a titre of 112 miu/ml, but surface antigen was not detectable, even after dissociation of antigen and antibody. As a result of this outbreak, 5 of the 11 boys remain carriers of the virus and 2 other family members have contracted acute hepatitis B. The possibility that the response of the haemophiliacs to HBV may be altered due to acquired alteration of their immune function is discussed. Regular screening of haemophiliacs, including those immune to hepatitis B, is recommended, since even with regular donor screening, HBV remains a major infective risk to haemophiliacs receiving Factor VIII replacement therapy, and the risk to an individual may change with time.     

A solid-phase enzyme immunoassay for the determination of IgM and IgG antibodies against translation products of pre-S1 and pre-S2 regions of hepatitis B virus

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.     

Genetic restriction of immune responsiveness to synthetic peptides corresponding to sequences in the pre-S region of the hepatitis B virus (HBV) envelope gene

Proteins of the HBV envelope (env) are coded for by two adjacent regions of the HBV env gene: the pre-S and S regions. Antigenic determinants corresponding to amino acid sequences of both regions are recognized by human antibodies and are important in virus-neutralizing responses. Protective immune responses to HBV appear to be linked to the major HLA histocompatibility complex. Inbred and congenic strains of mice represent a model system relevant for studies on the genetic control of immune responsiveness of humans to HBV envelope proteins. Such mouse strains were ranked according to their antibody response to the S protein and divided into high [d,q], intermediate [a,k,b], and low [s] responders (letters in brackets indicate H-2 haplotype.) Selected pre-S antigenic determinants can be mimicked with high fidelity by synthetic peptide analogues that are immunogenic without any carriers. Thus it is possible to study directly the genetic control of immune responsiveness to pre-S epitopes mimicked by these peptides without having to consider the influence of carriers or of S protein. The results presented here show that inbred mouse strains can be ranked according to their antibody responses to the synthetic peptide pre-S(120-145) as follows: A/J[a] approximately equal to SWR/J[q] greater than C57BL/6J[b] approximately equal to AKR/J[k] approximately equal to SJL/J[s] much greater than DBA/2J[d] greater than BALB/cJ[d]. Only SJL/J[s] mice responded well to another synthetic peptide pre-S (12-32). Thus, H-2-linked genes regulating the immune response to S protein and to epitopes on pre-S-coded sequences are distinct. Anti-pre-S(120-145) responses in S protein-nonresponders circumvent this nonresponsiveness. This should be considered in the design of hepatitis B vaccines.     

H-2 linked genetic control of immune responsiveness to hepatitis B surface antigen (HBsAg) in mice

Recent data suggest that genes involved in the control of (1) immune responses of humans to HBsAg and (2) the susceptibility to the development of chronic hepatitis B are linked to the major HLA histocompatibility complex. Studies on the genetic regulation of anti-HBs responses and on the possible abrogation of nonresponsiveness to HBsAg in humans are difficult. In an attempt to develop a relevant animal model system, the anti-HBs response of inbred and congenic strains of mice was investigated. A great variation in anti-HBs responses among individual mice belonging to the same strains was observed. Nevertheless, it was possible to rank the inbred mouse strains studied according to their decreasing anti-HBs responses as follows: BALB/c[d] ∽ SWR/J[q] > C57BL/6J[b] ∽ DBA/2J[a] > AKR/J[k] > A/J[a] > CBA/CaJ[k] > SJL/J[s]. (Letters in brackets indicate H-2 haplotype). Only a small proportion of SJL mice had an anti-HBs response. Therefore, this strain may serve as a model for human nonresponders. Studies with the congenic strains B10.D2[d] and B10.S[s] indicated that genes conferring responsiveness to HBsAg are linked to the H-2 histocompatibility complex. However, genes not linked to H-2 also probably play a role in regulating anti-Hbs responses.     

Production of hepatitis B virus surface antigen containing pre-S1 and pre-S2 domains by Chinese hamster ovary cells

Summary Cultured endothelial cells are shown to be induced in regard to permissiveness to human cytomegalovirus by temporary treatment postinfection with sodium butyrate (1–2mM). Drug-treated cells are demonstrated to exhibit expression of immediate early and early viral antigens, synthesis of viral DNA and viral structural glycoprotein B. Progeny virus could be visualized by electron microscopy.     

Expression of pre-S2 region of hepatitis B surface antigen in Escherichia coli

We constructed a recombinant plasmid that can express the entire pre-S2 sequence of hepatitis B surface antigen (HBsAg) as a fusion protein in E. coli. The hybrid protein, which comprises the bacterial TrpLE protein and the pre-S2 sequence, was the prominent protein that was found in cell extracts. As determined by immune blot analysis, this protein reacted with human HBV convalescent sera, as well as with sera from animals immunized with either purified HBsAg or isolated polypeptides containing pre-S2. It bound specifically to 125I-polymerized human albumin cross-linked with glutaraldehyde but not to 125I-monomeric human albumin. A novel adjuvant formulation was used in place of Freund's adjuvant to immunize guinea pigs with the recombinant product. The antisera obtained from serial bleedings were found to react with HBsAg of both d and y subtypes. These antisera were also shown to react solely with HBsAg polypeptides which contain of HBsAg to solid-phase polymerized the binding of HBsAg to solid-phase polymerized human albumin.