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1.
Yeast transposon of class-1 -based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications. 相似文献
2.
A plant-derived edible vaccine against hepatitis B virus. 总被引:52,自引:0,他引:52
J Kapusta A Modelska M Figlerowicz T Pniewski M Letellier O Lisowa V Yusibov H Koprowski A Plucienniczak A B Legocki 《FASEB journal》1999,13(13):1796-1799
The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein. 相似文献
3.
分泌型乙型肝炎病毒包膜M蛋白在毕赤酵母中的表达 总被引:1,自引:0,他引:1
将乙肝病毒包膜中蛋白M基因插入酵母整合型表达质粒pAO818的醇氧化酶 (AOX1)启动子下游 ,构建携带 8拷贝M表达盒的重组载体 ,经电穿孔转化SMD116 8菌株和G4 18筛选 ,得到了高效分泌表达M蛋白的毕赤酵母菌株 ,表达量超过 5 0mg L .经初步纯化 ,对表达产物的性质鉴定表明 ,重组蛋白具有preS2和S抗原性 ,可以形成颗粒 ,并具有一定程度的糖基化 .所构建的稳定重组菌株和所得到的重组蛋白颗粒 ,为进一步研究新一代的疫苗提供了必要的材料 . 相似文献
4.
Phenotypic mixing of rodent but not avian hepadnavirus surface proteins into human hepatitis B virus particles. 总被引:8,自引:4,他引:4
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The virus family Hepadnaviridae comprises two genera: orthohepadnaviruses isolated from humans (hepatitis B virus [HBV]) and rodents (e.g., woodchuck hepatitis virus [WHV]) and avihepadnaviruses isolated from birds (e.g., duck hepatitis B virus [DHBV]). They carry in their envelopes two (DHBV) or three (HBV and WHV) coterminal proteins referred to as small (S), middle (M), or large (L) surface protein. These proteins are also secreted from infected cells as subviral particles consisting of surface protein and lipid (e.g., 20-nm hepatitis B surface antigen for HBV). To investigate the assembly of these proteins, we asked whether surface proteins from different hepadnaviruses are able to mix phenotypically with each other. By coexpression and coimmunoprecipitation with species-specific antibodies, we could show the formation of mixed subviral particles and disulfide-linked heterodimers between the WHV S and HBV M proteins whereas the DHBV and HBV surface proteins did not coassemble. Complementation of HBV genomes defective in expressing the S or L protein and therefore incompetent to form virions was possible with the closely related WHV S protein or a WHV pre-S-HBV S chimera, respectively, but not with the less related DHBV S or L protein or with a DHBV L-HBV S chimera. The results suggest that the assembly of HBV subviral particles and virion envelopes requires relatively precise molecular interactions of their surface proteins, which are not conserved between the two hepadnavirus genera. This contrasts with the ability of, e.g., rhabdoviruses or retroviruses, to incorporate envelope proteins even from unrelated viruses. 相似文献
5.
Insertions in the hepatitis B surface antigen. Effect on assembly and secretion of 22-nm particles from mammalian cells 总被引:4,自引:0,他引:4
F Delpeyroux N Chenciner A Lim M Lambert Y Malpièce R E Streeck 《Journal of molecular biology》1987,195(2):343-350
The envelope protein of hepatitis B virus carrying the surface antigen, HBsAg, has the unique property of mobilizing cellular lipids into spherical or elongated particles, about 22 nm in diameter, which are secreted from mammalian cells. We have created mutant envelope proteins by insertion of various sequences of different lengths into two regions of the S gene encoding the major envelope protein. S genes carrying inserts in phase with HBsAg were expressed in mouse L cells from the simian virus 40 early promoter. Various single or double inserts in the two major hydrophilic domains of HBsAg were compatible with secretion of 22-nm particles. In all mutant envelope proteins studied, the HBsAg domains required for intracellular aggregation appeared to be intact. However, assembly into particles was not sufficient to assure transport into the extracellular space. The 22-nm HBsAg particle may be a useful vehicle for the export and presentation of foreign peptide sequences. 相似文献
6.
The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology. 相似文献
7.
Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles. 总被引:3,自引:3,他引:0
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Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible. 相似文献
8.
Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2. 总被引:22,自引:0,他引:22
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DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant. 相似文献
9.
Mina Nakauchi Hiroaki Kariwa Yasuhiro Kon Kentaro Yoshii Akihiko Maeda Ikuo Takashima 《Microbiology and immunology》2008,52(12):625-630
SARS‐CoV has four major structural proteins: the N, S, M, and E proteins. To investigate the mechanism of SARS‐CoV assembly, we cloned the genes encoding these four proteins into the eukaryotic expression vector pCAGGS and transfected them into 293T cells. When all four expression vectors were co‐transfected VLP formed, as confirmed using electron microscopy. Using a rabbit polyclonal antibody specific to the N protein, N‐protein‐containing particles similar in size to the VLP were also observed by immunoelectron microscopy, indicating that the VLP contained the N protein. Co‐immunoprecipitation analyses demonstrated an interaction between the N and M proteins, suggesting that N protein binds directly to M protein to be incorporated into VLP. 相似文献
10.
The middle hepatitis B virus envelope protein is not necessary for infectivity of hepatitis delta virus. 总被引:6,自引:5,他引:1
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The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte. 相似文献
11.
易于基因产物加工和快速纯化的融合表达载体 总被引:3,自引:0,他引:3
从Protein A基因酶切分离出编码完整的结合IgG的B、C区域(PABC)的相应DNA片段,克隆进噬菌体M1乳通过人工合成寡聚核苷酸引物,突变修饰B、C区域内分别含有的羟胺裂解位点,变As-Gly为Asn—A1a。利用突变修饰后的PBACm编码基因片段,构建了一组含有不同阅读框架的融合蛋白表达载体。进一步分别重组克隆了含人工合成的人胰岛索样生长因子Ⅰ(IGF—Ⅰ)、人和牛生长激素释放因子(GRF)等基因的表达质粒,在大肠杆菌中高效表达出PABC—IGF—l、PABC—hGRF、PABC-bGRF及其衍生型融合蛋白。经SDS—PAGE测定分析,每立升摇瓶的融合蛋白产量可达100mg以上。上述高效表达的PABC融合蛋白,通过固相亲和层析柱一步分离.获得SDS—PAGE鉴定为近均一分子量纯化的PABC融合表达产物。 相似文献
12.
Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus 总被引:2,自引:0,他引:2
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Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV. 相似文献
13.
Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles 总被引:10,自引:0,他引:10
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Balasuriya UB Heidner HW Hedges JF Williams JC Davis NL Johnston RE MacLachlan NJ 《Journal of virology》2000,74(22):10623-10630
RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies. 相似文献
14.
15.
S Kuroda S Otaka T Miyazaki M Nakao Y Fujisawa 《The Journal of biological chemistry》1992,267(3):1953-1961
The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen. 相似文献
16.
Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo. 总被引:4,自引:0,他引:4
M Levrero V Barban S Manteca A Ballay C Balsamo M L Avantaggiati G Natoli H Skellekens P Tiollais M Perricaudet 《Gene》1991,101(2):195-202
We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge. 相似文献
17.
Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain. 总被引:10,自引:3,他引:7
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Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes. 相似文献
18.
Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients
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Mancini-Bourgine M Bayard F Soussan P Deng Q Lone YC Kremsdorf D Michel ML 《Journal of virology》2007,81(10):4963-4972
Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression. 相似文献
19.
Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein. 总被引:1,自引:0,他引:1
The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes. 相似文献
20.
R Ulrich D Koletzki S Lachmann A Lundkvist A Zankl A Kazaks A Kurth H R Gelderblom G Borisova H Meisel D H Krüger 《Journal of biotechnology》1999,73(2-3):141-153
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. 相似文献