Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.     

Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity

The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.     

Immunogenicity of parenterally delivered plant-derived small and medium surface antigens of hepatitis B virus

Key message

Intramuscularly delivered plant-derived M-HBsAg was compared to S-HBsAg, and as a result elicited specific anti-preS2 antibodies and significantly higher titre of anti-HBs antibodies, together with IgG isotype profile indicating some Th1 polarisation, apart from the main Th2 response.

Abstract

HBV prevalence is still threatening, regardless of prevention programmes using vaccines containing S-HBsAg, supplemented by third-generation vaccines, comprising also M- and L-HBsAg. Plant expression systems offer a cost-effective production option of the antigens. Plant-derived S- and M-HBsAg, intramuscularly delivered to mice, elicited anti-HBs antibodies several times higher than high responsiveness threshold titre. M-HBsAg induced stronger response of anti-HBs and also specific anti-preS2 antibodies. IgG isotype profiles indicated mainly Th2 response, yet Th1 polarisation was also pointed out, in some larger extent for M-HBsAg. These results correspond to research on CHO-derived M-HBsAg vs. commercial vaccines based on S-HBsAg and support potency of plant-derived antigens as alternative injection vaccines.

     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

Overexpression of hepatitis B virus surface antigens including the preS1 region in a serum-free Chinese hamster ovary cell line

Current hepatitis B virus (HBV) vaccines consist of preparations of recombinant HBV major surface antigen (sAg) and are protective in about 90-95% of vaccinated subjects. In improved vaccines, the frequency of nonresponders to the classical vaccine could be reduced by including additional epitopes from the preS-domains of the middle and large surface antigens. In this report, the development and characterization of a CHO cell line for HBsAg, expressing major, middle, and large antigens are described. Despite the previously reported retention of secreted proteins by the preS1 domain, cell lines could be amplified that secreted large amounts of the complete set of antigens. A producer line was established that expressed 1mg HBsAg per 100ml suspension culture per week during exponential growth. The productivity per cell increased further by at least threefold when the culture reached the stationary phase at high cell densities. In the production cell line, several hundred copies of the HBV vector were integrated at two adjacent sites into chromosome 2. The cell line was adapted to growth in a defined protein-free medium minimizing the risk of adventitious agents introduced by animal derived supplements. The cell line stably produced antigen over several months. In the candidate vaccine, both preS2 and preS1 domains were present at ratios similar to HBsAg from human sera. In summary, a production cell line for an improved HBV vaccine is presented with properties such as high productivity, long term stability of expression, and growth in protein-free media.     

Inhibition of hepatitis B virus assembly with synthetic peptides derived from the viral surface and core antigens

The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.     

Antibody response to preS1 in hepatitis-B-virus-induced liver disease and after immunization

Antibodies to the preS1-encoded sequence of hepatitis B virus (HBV) envelope were detected by ELISA using a synthetic peptide analogue of preS1 proteins, in different groups of HBV-infected subjects and also in hepatitis B vaccine recipients. Such antibodies were specifically found in only 1 % of HBsAg chronic carriers including patients with cirrhosis and primary liver cancer. Anti-preS1 were detected in patients with acute hepatitis; in 13 % of the HBsAg⁺ sera obtained before recovery and in 37 % of the sera obtained after recovery.Anti-preS1 antibodies were detected in recipients of a plasma-derived vaccine, but not in those receiving a recombinant vaccine. The results indicate that anti-preS1 is an earlier serum marker of HBV clearance than anti-preS2 and anti-S antibodies.     

Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles

The long surface antigen polypeptide (L-HBsAg) of hepatitis B virus (HBV) is believed to mediate contact between the virus envelope and nucleocapsid protein (HBcAg). The N and C termini of L-HBsAg were shortened progressively in order to define the minimum contiguous sequence of amino acids that contains the residues necessary for association with HBcAg. The resulting mutants were expressed in rabbit reticulocyte lysates and their interaction with HBcAg was examined with an immunoprecipitation assay and an equilibrium binding assay in solution to give relative dissociation constants. Binding of HBcAg particles by L-HBsAg displayed two widely differing dissociation constants, indicating two distinct binding sites between the molecules. The two distinct sites, one located between residues 24 and 191 and the other between residues 191 and 322 of L-HBsAg, contribute synergistically to high-affinity binding to HBcAg, but disruption of either of these segments resulted in a much weaker interaction showing only one dissociation constant. Inhibition of the interaction by peptides that bind to the tips of the nucleocapsid spikes differentiated contacts in HBcAg for the two binding domains in L-HBsAg and implied that the amino-terminal binding domain contacts the tips of the HBcAg spikes. Analysis of specific single amino acid mutants of L-HBsAg showed that Arg92 played an important role in the interaction.     

Pharming vaccines for hepatitis and cytomegalovirus: Towards the development of multivalent and subunit vaccines for oral delivery of antigens

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30 nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.     

Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.     

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.     

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.     

HBV preS/S基因克隆及其在酿酒酵母中的表达

目的:探索利用酿酒酵母系统表达乙型肝炎病毒(HBV)preS／S基因。方法:利用PCR技 术,以HBV病毒DNA为模板,体外扩增HBV preS／S基因。然后构建重组表达载体pESC-preS／S。 用LiAc法转化酿酒酵母YPH50,选取重组菌进行培养,并诱导表达外源蛋白。提取蛋白浓缩后 进行SDS-PAGE分析,并经Western blot分析鉴定。结果:实验结果表明重组菌能够表达HBV preS／S蛋白。结论:利用酿酒酵母系统可成功表达HBV preS／S基因,为制备新型预防性疫苗提供 条件。     

修饰的含前S1、前S2免疫决定簇乙肝表面抗原融合多肽在毕赤酵母中的共表达

用BamHⅠ和BglⅡ双酶切含SS2嵌合基因片段的重组质粒pAO815-SS2,分离含AOX1启动子区、SS2嵌合基因和AOX1转录终止序列的表达盒。将分离的BamHⅠ-BglⅡ表达盒与经BamHⅠ线性化的pAO815-SS1质粒连接,转化大肠杆菌Top10F′,提取质粒,用BamHⅠ和BglⅡ双酶切分析重组质粒并筛选正向插入SS2表达盒的重组子。将该重组质粒用电转化法导入PichiapastorisGS115细胞,经表型筛选、小试表达和产物鉴定,构建了重组表达菌株GS115-SS1S2。重组菌株经甲醇诱导后制备抗原粗提液,进一步进行ELISA和Westernblot鉴定。ELISA结果显示,产物同时具有S、前S1和前S2抗原性。Westernblot分析进一步表明,表达产物既能与S抗体结合,也能与前S1和前S2抗体结合。工程菌经高密度发酵,表达量达到300~600mg/L。抗原经纯化后进行电镜观察,形成直径20~35nm的球形颗粒。纯化抗原经SDS-PAGE分析,SS1和SS2多肽仍然保持完整,基本无降解。     

O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection

BackgroundHepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg.MethodsHBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined.ResultsMass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(β1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro.ConclusionsGlycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection.General significanceThe detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.     

A tryptophan-rich motif in the carboxyl terminus of the small envelope protein of hepatitis B virus is central to the assembly of hepatitis delta virus particles

The small hepatitis B virus surface antigen (S-HBsAg) is capable of driving the assembly and secretion of hepatitis delta virus (HDV) particles by interacting with the HDV ribonucleoprotein (RNP). Previously, a specific domain of the S-HBsAg protein carboxyl terminus, including a tryptophan residue at position 196 (W196), was proven essential for HDV maturation (S. Jenna and C. Sureau, J. Virol. 73: 3351-3358, 1999). Mutation of W196 to phenylalanine (W196F) was permissive for HBV subviral particle (SVP) secretion but deleterious to HDV virion assembly. Here, the W196F S-HBsAg deficiency was assigned to a loss of its ability for interaction with the large HDV antigen (L-HDAg), a major component of the RNP. Because the overall S-HBsAg carboxyl terminus is particularly rich in tryptophan, an amino acid frequently involved in protein-protein interactions, site-directed mutagenesis was conducted to investigate the function of the S-HBsAg Trp-rich domain in HDV assembly. Single substitutions of tryptophan between positions 163 and 201 with alanine or phenylalanine were tolerated for SVP secretion, but those affecting W196, W199, and W201 were detrimental for HDV assembly. This was proven to result from a reduced capacity of the mutants for interaction with L-HDAg. In addition, a W196S S-HBsAg mutant, which has been described in HBV strains that arose in a few cases of lamivudine-treated HBV-infected patients, was deficient for HDV assembly as a consequence of its impaired capacity for interacting with L-HDAg. Interestingly, the fact that even the most conservative substitution of phenylalanine for tryptophan at positions 196, 199, or 201 was sufficient to ablate interaction of S-HBsAg with L-HDAg suggests that W196, W199, and W201 are located at a binding interface that is central to HDV maturation.     

Expression of the hepatitis B surface S and preS2 antigens in tubers of <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation. The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5 non-translated leader sequence, or the tuber-specific patatin promoter. HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters. The levels of preS2 antigen produced are the highest reported to date. Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.     

The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides—estimated at three to four per virion—is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S- and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.Hepatitis B virus (HBV) remains a major public health concern worldwide, affecting more than 350 millions of chronically infected individuals. Since the discovery of HBV, substantial information has been gathered on the viral replication cycle, but our understanding of the viral entry mechanism remains limited, and the identity of the receptor(s) for HBV is still unknown (15). HBV displays a very narrow host range, which is likely determined at viral entry by a highly specific interaction between the HBV envelope proteins and receptors at the surface of human hepatocytes. The envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (S-HBsAg) are membrane-spanning glycoproteins that differ from each other by the size of their N-terminal ectodomain (21). L-HBsAg contains a N-terminal pre-S1, central pre-S2, and C-terminal S domains. M-HBsAg is shorter than L-HBsAg in lacking pre-S1, whereas S-HBsAg consists of the S domain only (Fig. ​(Fig.1).1). Envelope protein synthesis occurs at the endoplasmic reticulum (ER) membrane. Empty subviral particles (SVPs) assemble from aggregates at a pre-Golgi membrane and exit the cell through the secretory pathway (36). Assembly of mature HBV virions requires, in addition to S-HBsAg, the presence of L-HBsAg as a matrix protein for nucleocapsid envelopment (6). Recent findings indicate that HBV virions and SVPs follow distinct pathways for budding: the late endosomal multivesicular bodies (MVBs) for HBV virions, and the MVB-independent secretory pathway for SVPs (26, 28, 46). The HBV envelope proteins can also package the hepatitis delta virus (HDV) ribonucleoprotein (RNP), in case of HBV/HDV coinfection (5, 45), leading to the formation of HDV virions. Whether HDV uses the SVP secretion pathway rather than an MVB-dependent route is uncertain.Open in a separate windowFIG. 1.Schematic representation of HBV envelope proteins. The topology of the L-, M-, and S-HBsAg proteins at the viral membrane is represented. The pre-S2 domain of L- and M-HBsAg, and the determinants of viral entry, pre-S1 and AGL, are indicated. The M-HBsAg protein, represented in gray, is dispensable for infectivity. The myristic acid (Myr) linked to the L-HBsAg N terminus is indicated (closed box). Subdomains 2-48 and 49-75 of the pre-S1 infectivity determinant are indicated. Open boxes represent transmembrane regions in the S domain.L-HBsAg, but not M-HBsAg, is crucial to infectivity of both HBV and HDV particles (13, 31, 41, 42). L-HBsAg contains a major infectivity determinant located between amino acid residues 2 and 75 of its N-terminal pre-S1 domain (4, 30), including a myristoyl anchor linked to glycine-2 (1, 8, 18), a putative receptor binding site (RBS) between positions 2 and 48, and a domain of unknown function between amino acids 49 and 75. To date, the most compelling evidence that pre-S1 mediates receptor binding comes from studies demonstrating that myristoylated synthetic peptides specific for the N-terminal 2-to-48 pre-S1 domain can bind to hepatocyte plasma membranes and block infection in vitro (3, 16, 17) and in vivo (37). Beside pre-S1, a second determinant was recently identified in the antigenic loop (AGL) borne by the three HBV envelope proteins (Fig. ​(Fig.1).1). The AGL participation in viral entry was first established in the HDV model (23) and more recently directly in the HBV model (39). Interestingly, serine substitutions for the AGL cysteine residues, which prove detrimental to the conserved immunodominant “a” determinant, could also block viral entry. Note that the “a” determinant consists in conformational epitopes, which elicit highly neutralizing antibodies (22). Infectivity and the “a” determinant were also lost when virions were treated with membrane-impermeable inhibitors of thiol/disulfide isomerization (2). These findings clearly established a correlation between the AGL cysteine disulfide bonds network, the conformation of the “a” determinant, and infectivity. Hence, the strict conservation of the “a” determinant among all HBV genotypes is related to the AGL function at viral entry. The AGL determinant may operate in association with, or independently of pre-S1, in binding to receptors at the early step of entry and/or in the mechanism of envelope disassembly postentry.In the present study, we investigated the pre-S1 determinant by performing transcomplementation experiments between mutants of 3 pre-S1 subelements: the myristoyl anchor, subdomain 2-48, and subdomain 49-75. We analyzed the activity of the AGL determinant in the S- or L-HBsAg background (S- and L-AGL, respectively), and we examined the effect of introducing increasing amounts of infectivity-deficient pre-S1, or AGL, in the virion''s envelope on infectivity.     

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.