Characterization of complex B cell epitopes on woodchuck hepatitis virus surface antigens by using plasmids encoding chimeric proteins and DNA immunization

The conformational nature of the B cell epitopes on the hepadnavirus surface antigens makes its characterization difficult. Here, a new approach by DNA vaccination with plasmids expressing chimeric hepadnavirus surface antigens was explored to determine B cell epitopes on the surface antigens of woodchuck hepatitis virus (WHsAg). A series of chimeric genes consisting of complementary fragments of WHsAg and hepatitis B virus surface antigens (HBsAg) was constructed. These plasmids expressed the following: (i) middle chimeric surface antigens (MCSAgs), including pre-S2 region and small surface antigens; (ii) small chimeric surface antigens (CSAgs); (iii) a mutated WHsAg with two amino acid substitutions, the Leu 136 to Thr and Ala 140 to Asp, within the central immunogenic region. The mutated region from amino acid 135 to 143 within WHsAg mimics the second loop of the HBsAg a-determinant. MCSAgs and CSAgs were expressed in transiently transfected mammalian cells and were reactive to anti-HBsAg and anti-WHsAg, as shown by indirect immunofluorescence staining and ELISA. Vaccination with plasmids encoding MCSAgs induced strong antibody responses to the pre-S2 region. Anti-pre-S2 antibodies were directed to a linear, immunodominant region within the amino-terminal region of the pre-S2 region and were able to precipitate serum WHsAg. Vaccinations with the plasmids expressing the CSAgs led to the conclusion that an extended region aa 116-169 of WHsAg, analogous to the HBsAg a-determinant, was sufficient for the induction of anti-WHsAg antibodies. The mutated WHsAg with the second loop of the HBsAg a-determinant efficiently induced anti-WHsAg antibodies, but also a low titer of anti-HBsAg. Thus, multiple B cell epitopes of a linear and conformational nature are present on WHsAg. We presented an efficient and broadly applicable strategy for analysis of complex immunogenic determinants of natural or mutated viral antigens.     

Impact of amino acid sequence variation of a determinant(s) on the antigenic properties of hepatitis B virus HBsAg

Impact of amino acid sequence variation on the antigenic properties of the surface hepatitis B virus antigen HBsAg was studied. Eleven recombinant HBsAg variants of wild (adr, ayw2, adw2, adw4, aywl, adw2) and vaccine escape (adw2 T126S, adw2 Q129L, adw2 Q129R, adw2 T143K, adw2 Q145R, aywl Q145A) were obtained. All the recombinant antigens were tested on a panel of 43 monoclonal antibodies (MAb) specific to different HBsAg determinants. Amino acid sequence variation of the a-determinant of HBsAg was shown to significantly affect its immunological responsiveness and antigenic properties. Amino acid substitution in different positions or in the same position, but for various amino acids may differently affect these properties.     

A study of the antigenicity and immunogenicity of a new hepatitis B vaccine using a panel of monoclonal antibodies

The successful prevention of infection with hepatitis B virus (HBV) has been achieved by vaccination with purified hepatitis B surface antigen (HBsAg). The ability of a novel synthetic HBV envelope antigen vaccine (Hep B-3, Hepagene ™; Medeva), which contains part of the pre-S1 and the complete pre-S2 regions and the whole of the S region and was produced in a mammalian cell line, to induce antibodies required for a protective immune response is of importance. In this study, the use of a panel of monoclonal antibodies known to bind to epitopes within the common “a” determinant has demonstrated that the epitopes present on this new vaccine are comparable to those found with plasma-derived HBsAg. In addition, the epitope specificity of the antibodies induced by this vaccine was examined and shown to accord well with previous results obtained using both a plasma-derived vaccine and a recombinant vaccine prepared in yeast. J. Med. Virol. 54:1–6, 1998. © 1998 Wiley-Liss, Inc.     

Cellular immunity to nucleocapsid and pre-S determinants in asymptomatic carriers of hepatitis B virus.

Previous studies of cellular immunity in asymptomatic HBV carriers have been limited to evaluation of responses to plasma-derived HBsAg preparations. We have explored the specificity of cellular immune responses to HBV antigens in these subjects using an indirect T-lymphocyte migration inhibitory factor assay and three antigen preparations (recombinant nucleocapsid antigen (HBcAg), plasma-derived HBsAg with or without pre-S2, and Saccharomyces cerevisiae-synthesized HBsAg without pre-S2 region). T cells from 10 asymptomatic chronic HBV carriers with normal liver function tests were responsive to nucleocapside determinants (mean migration index = 0.55 +/- SD 0.07) and to pre-S2-positive plasma-derived HBsAg (MI = 0.62 +/- 0.05). However, none responded to HBsAg devoid of pre-S2 sequences (MI = 0.98 +/- 0.04). In further experiments, T cells from three HBV carriers, cultured with six different HBsAg preparations, exhibited responsiveness only to those preparations containing significant pre-S2 activities. Our results show that T-cell immunity to nucleocapsid determinants of the virus and HBsAg in present in asymptomatic HBV carriers; the latter is restricted to antigenic preparations containing significant pre-S2 activities. Hence, T-cell immunity to pre-S determinants may not always be associated with HBV clearance.     

Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein

Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.     

Immunity to hepatitis B: analysis of antibody and cellular responses in recipients of a plasma-derived vaccine using synthetic peptides mimicking S and pre-S regions.

The potential of a panel of synthetic HBsAg peptides as components of a synthetic hepatitis B vaccine was assessed. Each was used in turn as probes to analyse human immune responses to a licensed plasma-derived HBV vaccine. Both humoral and cellular responses were analysed with synthetic peptides representing residues 124-147 of the surface antigen of the virus (HBsAg) and residues 126-140 of the pre-S2 region. Antibody levels and affinities were assessed in radioimmunoassays with synthetic linear and cyclical forms of surface antigen peptides 124-137 and 139-147, with the gp30p25 polypeptide complex of HBsAg and with the linear pre-S2 peptide 126-140. Levels and affinities of antibodies to the antigens increased with time during immunization. However, antibodies binding the cyclical peptide representing amino acids 139 to 147 (C139) were present at higher levels and had higher affinities than were antibodies binding the other peptides, indicating that C139 more closely approximates a domain on the native antigen than do the other peptides. No humoral responses were measured with the pre-S2 peptide. Cellular responses were assessed by in vitro stimulation of peripheral blood lymphocytes by HBsAg and by the synthetic peptides. All vaccine recipients had demonstrable lymphocyte responsiveness to HBsAg after both second and third doses of the vaccine. Of the S and pre-S peptides used, only L124 failed to induce lymphocyte stimulation in all recipients. However, there were individual variations in both the time of initial responsiveness to peptides and in the level and time of maximal stimulation. Stimulation by native HBsAg particles, which corresponded to the appearance of anti-HBs antibody, preceded that observed using synthetic peptides. In all recipients, maximum stimulation indices with HBsAg were significantly higher than those observed with the peptides. In contrast to the absence of pre-S2 antibody, the lymphocytes from all recipients showed positive stimulation in response to the peptide representing residues 126-140 of the pre-S2 region. None of these individuals had antibodies to pre-S or an HB core peptide sequence nor did their lymphocytes respond to a synthetic peptide representing an HB core sequence.     

Expression of hepatitis B virus antigens in attenuated Salmonellae for oral immunization

The aim of our work is to identify hepatitis B virus antigens that can be stably expressed in attenuated Salmonellae and elicit protective immune responses as live oral route vaccines. As a first carrier system, we expressed T-cell and B-cell epitopes of hepatitis B virus as fusion proteins with the non-toxic subunit B (LT-B) in attenuated Salmonellae. These recombinant Salmonellae elicited anti-LT-B T- and B-cell immune responses and anti-HBV nucleocapsid antigen (HBcAg) T-cell responses when fed to mice. To combine the protective potential and the high immunogenicity of HBc with the induction of virus neutralizing antibodies to HBV surface antigen, we constructed vectors expressing hybrid HBc/pre-S particles in which the pre-S epitopes were surface-exposed. With one of these vectors, stable constitutive high level expression of hybrid HBc/pre-S2 particles was achieved in several attenuated Salmonella strains. When recombinant Salmonellae expressing such hybrid HBc/pre-S2 fusion proteins were fed to mice, the animals developed high titres of anti-HBcAg-specific serum IgG after a single or multiple oral immunizations, depending on the strain used as a carrier. In addition, lower titered antibodies against the pre-S2 antibody-binding sites were elicited. This is the first HBV antigen eliciting high-titered immune responses after a single oral immunization in recombinant Salmonellae. The immunogenicity of periplasmic LT-B and cytoplasmic HBc/pre-S2 shows that surface exposure of a foreign antigen is not a prerequisite for its immunogenicity in live attenuated Salmonellae.     

Analysis of the antigenic epitopes of hepatitis B surface antigen involved in the induction of a protective antibody response.

Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.     

High-level expression and characterization of hepatitis B virus surface antigen in silkworm using a baculovirus vector.

The Bombyx mori nuclear polyhedrosis virus (BmNPV) in the silkworm was used successfully for mass production of biologically active foreign genes under the control of the polyhedrin promoter. This system was adapted for the production of large amounts of hepatitis B virus surface antigens (HBsAg). The DNA fragments coded for the middle protein, which is composed of the S protein with the pre-S2 region, were cloned, the signal protein gene of beta-IFN was added, and both were inserted into a cloning vector. After co-transfection with wild-type BmNPV, stable recombinant viruses were isolated by the limiting dilution method. Infected silkworm larvae with the recombinants expressed HBsAg at high levels (400-600 micrograms/ml). These products, consisting of two polypeptides with molecular weights of approximately 25,000 (p25) and glycosylated P25 (GP28), were purified as assembled 22-nm particles. We demonstrated that HBsAg from silkworms consists of S protein with 7 amino acids of Pre-S2.     

Monoclonal antibody recognizing pre-S(2) epitope of hepatitis B virus: characterization of pre-S(2) epitope and anti-pre-S(2) antibody

A hybrid cell line producing monoclonal antibodies recognizing an epitope encoded by the pre-(S)2 region of hepatitis B virus (HBV) genome was obtained by fusion of mouse myeloma cells with lymphocytes from mice immunized with HBV. The monoclonal antibody Mo-F124 secreted from the hybrid line reacted with the pre-S(2) epitope expressed on the surface of both viral and recombinant HBsAg particles--pre-S(2) and S gene product--localised on 34 kD glycoprotein of the viral envelope. The pre-S(2) epitope was sensitive to digestion with V8 protease from Staphylococcus aureus. The enzyme abolished reactivity with Mo-F124 and polymerized human serum albumin (pHSA) binding activity of recombinant particles. Mo-F124 antibody was used to develop highly sensitive radioimmunoassays for determination of pre-S(2) epitope and anti-pre-S(2) antibody in sera of hepatitis B patients. Detection of a pre-S(2) epitope by the monoclonal antibody-based assay in the early phase of acute HBV infection correlated well with the presence of markers of active viral replication (HBeAg, HBV DNA). The appearance of anti-pre-S(2) antibody, usually in the third month after onset of symptoms, was followed by elimination of circulating HBsAg and seroconversion to anti-HBs in all tested cases of uncomplicated acute hepatitis followed by recovery. Anti-pre-S(2) response was not observed in patients with chronic hepatitis B or acute HBV infection progressing to chronic disease. The observed correlation of anti-pre-S(2) response with recovery suggests that the pre-S(2) epitope may represent one of the epitopes inducing antibodies that neutralize the hepatitis B virus.     

Occurrence of pre-S1 antigen in viremic and nonviremic carriers of hepatitis B surface antigen

The proteins of viral envelope, encoded by the pre-S1 region of HBV-DNA, were measured quantitatively with enzyme immunoassay using monoclonal antibodies directed to pre-S1 epitope and correlated with the expression of pre-S2 region encoded epitope and other HBV markers. In acute HBV infection, both pre-S encoded proteins were detected in sera along with markers of viral replication and disappeared shortly before complete virus clearance while high HBsAg titers were still present. Pre-S1 antigen was present in most (95.5%) symptomatic and asymptomatic chronic HBsAg carriers. There was no correlation between the presence of pre-S1 and HBeAg or HBV-DNA in serum: 73% of sera with pre-S1 determinants were anti-HBe positive, and only 25.4% were positive for HBV-DNA. Most pre-S1 activity in sera of viremic carriers was detected in fractions of sucrose gradient containing subviral 22-nm particles, and much less in those containing infectious virions. In asymptomatic, nonviremic HBsAg carriers, pre-S1 was located only on subviral 22-nm forms. Pre-S1 positive particles had no accessible pre-S2 epitope, which is recognized specifically by monoclonal anti-pre-S2 (F124) antibody. These results show that the synthesis of the large protein of HBV envelope may occur also in the absence of active viral replication, and in these cases pre-S1 encoded sequences are on subviral particles of HBsAg. Therefore, pre-S1 is not a serologic marker of infectious virus. Disappearance of pre-S1 epitopes on HBsAg occurs only before complete clearance of the virus, and this may have potential prognostic relevance.     

Detection of hepatitis B virus pre-S1 and pre-S2 determinants in paraffin wax embedded liver tissue: importance of reagents used.

The presence of pre-S polypeptides in paraffin wax embedded liver sections from the biopsy specimens of 15 hepatitis B surface antigen (HBsAg) seropositive patients (five with chronic persistent hepatitis (CPH), four with chronic active hepatitis (CAH), four with cirrhosis and two "healthy" HBsAg carriers) was investigated using monoclonal antibodies directed to distinct epitopes on pre-S1 (18/7 and TO 606) and pre-S2 (5535 and Q 19/10). Pre-S1 was found in 13 cases when MA 18/7 was used but in only one specimen when TO 606 was used. Pre-S2 was detected in all the biopsy specimens with 5535 and in eight samples with Q 19/10. Mild enzymatic digestion annulled the staining of all monoclonal antibodies but Q 19/10. No association was observed between pre-S polypeptide expression and hepatitis B virus (HBV) replication or disease severity. Pre-S polypeptides can be detected readily in paraffin wax embedded material but the results obtained largely depend on the monoclonal antibodies used.     

Overlapping gene mutations of hepatitis B virus in a chronic hepatitis B patient with hepatitis B surface antigen loss during lamivudine therapy

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yrold Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.     

New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection.

We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.     

A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.     

Igh allotype-linked control of immune complex-type hypersensitivity induced by hepatitis B surface antigen.

Hepatitis B surface antigen (HBsAg) induced an immune complex-type hypersensitivity which developed at 5-6 hr after the challenge and could be transferred by anti-HBs antisera, in addition to the delayed-type hypersensitivity in B10.BR mice. The pre-S region of HBsAg influenced this induction because the pre-S-depleted HBsAg or recombinant major S protein could not stimulate this 5-hr ear swelling. The male B10.BR mice responded better than the female ones, probably due to the inhibition by female hormone. Furthermore, HBsAg could also induce an early-occurring hypersensitivity which appeared within 1 hr of the antigen challenge. However, B10.BR mice did not exhibit this hypersensitivity; B6 mice expressed all three types of hypersensitivity (1 hr, 5 hr and 24 hr) and BALB/c mice showed only 1-hr and 24-hr responses. The expression of the immune complex-type seems to be determined by the Ighb gene or gene(s) closely associated to it. Mice bearing the Igh-1b allotype could be stimulated by HBsAg to induce the immune-complex type hypersensitivity. Moreover, it was the high specific binding not the titre of anti-HBs antibodies that influenced the exhibition of immune complex-type hypersensitivity.     

Enzyme-linked immunoassay of pre-S gene-coded sequences in hepatitis B vaccines

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.     

Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus

Summary Seven fragments of the spike (S) gene cDNA of transmissible gastroenteritis virus (TGEV), as well as the full length cDNA, were cloned and expressed in baculovirus vectors. Piglets were immunized with cells infected with the recombinant viruses. Each of the recombinants induced TGEV-specific antibodies detected in a fixed cell enzyme immunoassay. The amino terminal half of the S protein, containing all four major antigenic sites (A, B, C and D), and encoded by a 2.2 kb fragment of the S gene, induced virus neutralizing (VN) antibody titers comparable with those induced by the complete S protein. Recombinant proteins lacking the A antigenic site, or with a deletion including the putative receptor binding sites and the D antigenic site, were not capable of inducing levels of VN antibodies similar to those induced by the whole S protein.     

Liposome-entrapped T-cell peptide provides help for a co-entrapped B-cell peptide to overcome genetic restriction in mice and induce immunological memory.

We have investigated the possibility of a T-cell epitope peptide providing help for a B-cell epitope peptide when both peptides are co-entrapped in the same liposomes. Epitope models used were a 28 amino acid peptide from the S region of the hepatitis B surface antigen (HBsAg) (subtype adw) containing an H-2s Th-cell epitope, and a 33 amino acid peptide from the pre-S1 region of the HBsAg (subtype adw) designed to exclude an adjacent H-2s T-cell epitope, the latter (pre-S1) peptide being recognized by SJL (H-2s) mice as a B-cell epitope. SJL(H-2s) mice were immunized twice intramuscularly with S or pre-S1 peptide alone, co-entrapped in the same liposomes or entrapped in separate liposomes which were mixed before injection. Analysis of sera for anti-peptide IgG1 antibodies revealed that the Th-cell peptide provided help for the pre-S1 peptide only when the two peptides were co-entrapped in the same vesicles. This helper effect was found to correlate with the ability of S peptide (co-entrapped with the pre-S1) to stimulate T-cell proliferation in vitro. There was no IgG1 response against pre-S1 peptide in mice immunized with a mixture of the free peptides or a mixture of separately entrapped peptides. A helper effect, albeit much weaker, was also observed in mice immunized with the two peptides emulsified in incomplete Freund's adjuvant. Antisera from mice immunized with both peptides co-entrapped in liposomes were found to bind to full length (pre-S1 containing) recombinant HBsAg. Moreover, binding values were much higher than those seen with antisera from animals immunized with the liposomal S peptide above, presumably because of full access of anti-pre-S1 antibodies to the pre-S1 region of the rHBsAg. It is concluded that liposomes could serve not only as an immunological adjuvant for peptides but also as a carrier for Th- and B-cell epitopes thus eliminating the need for covalent linkage to a carrier protein.