Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein

Abstract Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc_3–144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc_1–183) could not be purified or characterised immunologically, although it formed core like particles. HBc_3–144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7–17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc_3–144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.     

Behavior of a short preS1 epitope on the surface of hepatitis B core particles

The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.     

Comparative immunogenicity of hepatitis B virus core and E antigens

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.     

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.     

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid.     

Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface

Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells. These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg.     

Combinatorial approach to hepadnavirus-like particle vaccine design

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the "preexisting immunity" and "assembly" problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.     

T- and B-lymphocyte responses to human immunodeficiency virus (HIV) type 1 in macaques immunized with hybrid HIV/hepatitis B surface antigen particles.

Recombinant human immunodeficiency virus (HIV)/hepatitis B surface antigen (HBsAg) subviral particles of dual antigenicity and immunogenicity were obtained by fusing 84 amino acids of the HIV type 1 external envelope glycoprotein within the pre-S2 part of the hepatitis B middle protein (M.-L. Michel, M. Mancini, E. Sobczak, V. Favier, D. Guétard, E.-M. Bahraoui, and P. Tiollais, Proc. Natl. Acad. Sci. USA 85:7957-7961, 1988). We now describe the humoral and cellular immune response of rhesus monkeys immunized with these hybrid particles. Macaque antisera raised by subcutaneous injections of the HIV/HBsAg particles were shown to be specific for HIV in peptide-binding assays. Moreover, we were able to generate in these vaccinated animals a T-cell-proliferative response to both parts of the hybrid particle, i.e., HIV and HBsAg. These results establish the presence of a T-cell epitope in this HIV segment, which has been shown previously (L.A. Lasky, G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon, Cell 50:975-985, 1987) to be an important domain involved in the binding of the virus to its cellular receptor, the CD4 molecule. This work demonstrates the feasibility of using the HBsAg subviral particle as a carrier protein for the presentation of foreign immunogenic epitopes to the immune system.     

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.     

Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons.

Two recombinant plasmids were constructed that allow expression of the hepatitis B core (HBc) antigen gene in the yeast Saccharomyces cerevisiae under the control of the repressible acid phosphatase promoter. One plasmid was designed to produce polypeptide I, which consists of 183 amino acids, and the other plasmid was designed to produce polypeptide II, which has an additional 29-amino-acid sequence at the amino terminus of polypeptide I. The viral genome may code for either one or both of these two polypeptides, depending upon the selection of initiation codons. Both polypeptides produced in yeast cells reacted with anti-HBc antibody and were assembled into spherical particles approximately 27 nm in diameter. Particles made of polypeptide I were stable, whereas those made of polypeptide II readily dissociated when exposed to high salt levels. The antigenicity of the HBc (as defined by its reactivity to anti-HBc antibody in the reversed passive hemagglutination assay) disappeared as the particle dissociated, leaving materials that sedimented slowly and that reacted to anti-hepatitis B e antibody. These observations strongly suggest that native viral cores are mostly (if not all) made of polypeptide I, because it is reasonably stable, and that the N-terminal portion of this polypeptide has some, but not a profound, influence on the assembly of polypeptides into particles.     

High-affinity interactions between peptides and heat shock protein 70 augment CD8+ T lymphocyte immune responses

Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-K(b) OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.     

Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes.

We previously determined a number of 'permissive' sites in the periplasmic maltose-binding protein (MalE) from Escherichia coli. These sites accept the insertion of heterologous peptides without major deleterious consequences for the activities, structure and cellular location of the protein. This study explores the versatility of two such permissive sites for the synthesis of foreign peptides, and examines the antigenicity and the immunogenicity of the inserts. One site is located after amino acid 133 (aa133) of MalE, and the other after aa303. Both sites tolerate inserts of up to at least 70 aa and accept sequences of different natures. Hydrophobic aa sequences are accepted, although strongly hydrophobic sequences, such as the Sendai virus F protein membrane anchor, affected export. We compared the antigenic and the immunogenic properties of peptides derived from the coat proteins of HBV and poliovirus which contain well defined B-cell epitopes. Specific monoclonal antibodies show that the antigenic properties of the inserted B-cell epitopes were different at the two sites. Despite these differences, the inserted peptides elicited strong and comparable antibody responses in mice against the corresponding synthetic peptides. In this case, and with these criteria, the molecular context of the peptides did not affect the immunogenicity of B-cell epitopes. We show for the first time that when a foreign peptide carrying a T-cell epitope was inserted in MalE, the hybrid proteins can elicit a T-cell response against the foreign peptide both in vivo and in vitro. Furthermore, the MalE hybrid was as efficient as free peptide in stimulating T-cell hybridomas in vitro. The MalE vectors provide a powerful genetic system to study how the position and the conformation of a peptide within a protein affect the B-cell and T-cell responses.     

A new unconventional HLA-A2-restricted epitope from HBV core protein elicits antiviral cytotoxic T lymphocytes

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141–149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLAA2 transgenic mice. Finally, we show that mutations in HBc141–149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.     

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.     

The core antigen of hepatitis B virus as a carrier for immunogenic peptides

The core antigen of hepatitis B virus (HBcAg) made in Escherichia coli yields particles that closely resemble the viral nucleocapsid. Extensive modifications can be made to the primary structure of HBcAg without impairing particle assembly. This enables other peptide sequences, including very long sequences, to be added, substituted, or inserted into the nucleocapsid subunit while retaining the ability to form highly immunogenic particles. These also retain the T cell epitopes of HBcAg and constitute powerful delivery systems for a diverse range of immunogenic epitopes and have significant potential for development of multicomponent vaccines.     

乙肝前S2抗原决定簇串联体与核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇,为增强ＰｒｅＳ２的抗原性,本实验采用将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因以串联方式与ＨＢｃＡｇ的基因进行了融合,融合基因在大肠杆菌中获得表达,并通过ＥＬＩＳＡ比较研究了融合蛋白中ＰｒｅＳ_２ｅｐｉｔｏｐｅ单体及串联体的抗原性差异。     

Enhancing the immunogenicity of a permissive binding T cell epitope derived from the simian immunodeficiency virus-encoded negative regulatory factor.

Using the murine system we have analyzed an immunogenic T cell peptide epitope corresponding to amino acids 96-112 of the simian immunodeficiency virus-negative regulatory protein sequence. This epitope was unusual in that it was strongly immunogenic in mice of five of the six H-2 haplotypes tested. We generated a T cell hybridoma (SVNF) specific for this peptide in order to determine how manipulating the peptide might alter its immunogenicity. Substitution analysis showed that His 103, Pro 104, Val 106, and Pro 107 were important amino acids for stimulating SVNF because substitutions at these positions diminished the reactivity of SVNF. However, we also found that substituting an Ala for a Val at position 100 or a Val for an Ala at position 110 enhanced reactivity of SVNF. We were able to further enhance the immunogenicity of this epitope by extending the carboxyl terminus two amino acids and making the resulting carboxyl-terminal Lys an amide and by adding a Glu to the amino terminus. These modifications shifted the in vitro activity of SVNF at least two orders of magnitude. We also compared the ability of this modified peptide and the wild-type SIV nef 96-112 to prime a T cell response in vivo. We primed mice with various doses of either the wild-type or the modified peptide and looked at the ability of the draining lymph node cells to proliferate to wild-type peptide. We found that the modified peptide was 10- to 100-fold better at priming a T cell response than the wild-type peptide. Therefore, we were able to create a peptide that was more immunogenic than the wild-type peptide in vivo as well as in vitro. Manipulations such as these that enhance the immunogenicity of T cell epitopes must be considered in developing peptide vaccines against HIV or other infectious agents.     

Molecular Basis for the Interaction of the Hepatitis B Virus Core Antigen with the Surface Immunoglobulin Receptor on Naive B Cells

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.     

Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.