Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.     

Thermal stability of hepatitis B surface antigen S proteins.

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.     

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.     

Organ-specific expression of hepatitis B surface antigen in potato

Summary The gene encoding the hepatitis B surface antigen (HBsAg) under the control of cauliflower mosaic virus 35S-promoter was constructed and expressed in transgenic potato plants. The HBsAg expression were measured by ELISA kit containing monoclonal antibodies. The amount of HBsAg in roots was found 5–10 fold higher than in leaf tissues     

Epitopes associated with a synthetic hepatitis B surface antigen peptide

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 µg/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.     

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.     

Hepatitis B surface antigen. Role of lipids in maintaining the structural and antigenic properties of protein components.

Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.     

Antigenic structure of hepatitis B surface antigen: identification of the "d" subtype determinant by chemical modification and use of monoclonal antibodies

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes were reductively methylated with formaldehyde in the presence of sodium cyanoborohydride. The effect on antigenicity was determined by radioimmunoassay with monoclonal antibodies specific for seven different antigenic determinants. The reaction was shown to eliminate specifically the "d" antigenic activity of HBsAg/adw and to have no effect on HBsAg/ayw. Moreover, the reaction had only a slight affect on HBsAg/adw at one of the "a" antigenic determinants. The sites of modification were determined and the extent of modification of each site was compared to the loss of "d" antigenic activity. These studies demonstrated that the loss of "d" activity was due to the modification of lysine 122 in HBsAg/adw, and that although the amino terminus and lysine residues 141 and 160 of both HBsAg/adw and HBsAg/ayw are reactive, their modification does not alter any measurable antigenic activity.     

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies

Summary SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG_1, isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1–126 and 49–149 but failed to bind to fragment 99–149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.     

Immunological analysis of chemoattractive proteins from earthworm to garter snakes.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.     

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

Expression vectors for the construction of hybrid Ty-VLPs

Purification of expressed proteins can be facilitated by expressing the rccombinant protein as a fusion with a carrier protein that assembles into paniculate structures. This article describes the use of expression vectors in producing a hybrid of the yeast retrotransposon Ty, which self-assembles into virus-like particles (VLPs). Hybrid VLPs can be used in such laboratory applications as the production of polyclonal and monoclonal antibodies, structure/function analyses, the detection of important antigenic determinants, and cpitope mapping of monoclonal antibodies.     

Isolation and characterization of monoclonal antibodies against ribonuclease inhibitor

Mouse monoclonal antibodies were generated against human ribonuclease inhibitor, an intracellular regulatory protein. A total of four antibodies were isolated, all of which were of the immunoglobulin G1 subtype. Western blot analysis of the antibodies suggested monospecificity. Based on immunoradiometric competition assays two of the antibodies were determined to be directed against the same antigenic epitope, while the other two were against a second and possibly third epitope. None of the antibodies appeared to be directed against the ribonuclease binding site of the antigen. Data is presented suggesting that ribonuclease inhibitor is present in normal human serum. The potential significance of these findings is discussed.     

Properties of monoclonal antibodies interacting with determinants of hepatitis B virus surface antigen

Fourteen hybridoma clones have been isolated producing the monoclonal antibodies to the surface antigen of the hepatitis B virus (HBsAg). Monoclonal antibodies have been shown to react in high titres with HBsAg in the reactions of PHA, PH and ELISA. The specificity of monoclonal antibodies to two antigenic determinants has been found by the competitive solid phase ELISA technique. Monoclonal antibodies from nine clones react with one determinant while monoclonal antibodies from the rest five clones react with the other nonoverlapping determinant.     

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.     

Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.     

Antibodies recognizing a variety of different structural motifs on meningococcal Lip antigen fail to demonstrate bactericidal activity.

The neisserial Lip antigen is a conserved antigen associated with the pathogenic Neisseria species, and is composed of multiple repeats of a consensus pentapeptide. A series of monoclonal antibodies reacting with meningococcal Lip antigen were subjected to epitope mapping, using solid-phase synthetic peptides based on the consensus repeat sequence. The antibodies were found to recognize different continuous epitopes based on the consensus sequence. One monoclonal antibody was utilized in affinity chromatography to obtain purified Lip antigen and the antigen was used for immunization of mice. The resulting antisera did not recognize Lip antigen on Western blots but reacted specifically with Lip antigen in immune precipitation experiments, indicating that the predominant polyclonal immune response was directed against conformational epitopes. Despite the diversity of both continuous and conformational epitopes recognized by the antibodies produced, none of the antibodies demonstrated the ability to promote complement-mediated bactericidal activity. Thus despite its initial apparent promise as a potential vaccine candidate the case for the inclusion of Lip antigen in vaccine formulation cannot be supported at present.