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1.
Miao Li Yu-Fei Wang Yu Wang Hui Gao Na Li Yuan Sun Bing-Bing Liang Hua-Ji Qiu 《Immunology letters》2009,125(2):145-150
Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections. 相似文献
2.
Characterization of antibody responses against a neutralizing epitope on the glycoprotein E2 of classical swine fever virus 总被引:1,自引:0,他引:1
The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies. 相似文献
3.
Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus 总被引:2,自引:0,他引:2
Risatti GR Holinka LG Carrillo C Kutish GF Lu Z Tulman ER Sainz IF Borca MV 《Virology》2006,355(1):94-101
Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). In vitro growth of mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) was similar to parental BICv, while mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence. 相似文献
4.
Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine 总被引:4,自引:0,他引:4
Risatti GR Holinka LG Lu Z Kutish GF Tulman ER French RA Sur JH Rock DL Borca MV 《Virology》2005,343(1):116-127
Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics in primary porcine macrophage cell cultures, the major target cell of CSFV in vivo, it was markedly attenuated in swine. All RB-C22v-infected pigs survived infection remaining clinically normal in contrast to the 100% mortality observed for BICv-infected animals. Comparative pathogenesis studies demonstrated a delay in RB-C22v spread to, and decreased replication in the tonsils, a 10(2) to 10(7) log10 reduction in virus titers in lymphoid tissues and blood, and an overall delay in generalization of infection relative to BICv. Notably, RB-C22v-infected animals were protected from clinical disease when challenged with pathogenic BICv at 3, 5, 7, and 21 days post-RB-C22v inoculation. Viremia, viral replication in tissues, and oronasal shedding were reduced in animals challenged at 7 and 21 DPI. Notably BICv-specific RNA was not detected in tonsils of challenged animals. These results indicate that a carboxyl-terminal domain of E1 glycoprotein affects virulence of CSFV in swine, and they demonstrate that mutation of this domain provides the basis for a rationally designed and efficacious live-attenuated CSF vaccine. 相似文献
5.
The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2 总被引:1,自引:0,他引:1
Liu S Tu C Wang C Yu X Wu J Guo S Shao M Gong Q Zhu Q Kong X 《Journal of virological methods》2006,134(1-2):125-129
Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829-837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al., two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828-842 amino acids of E2. The gene cassette was fused in-frame to 3' terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-beta-d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV. 相似文献
6.
Krivonos AV Zaberezhny AD Grebennikova TV Gibadulin RA Musienko MI Tsibezov VV Bogdanova VS Kal'nov SL Aliper TI Nepoklonov EA 《Voprosy virusologii》2000,45(2):29-36
Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay. 相似文献
7.
Protection of mice against lethal challenge with herpes simplex virus by vaccination with an adenovirus vector expressing HSV glycoprotein B 总被引:11,自引:0,他引:11
Increasing attention has been focused on the use of recombinant mammalian viruses as potential vaccines. Recombinant human adenoviruses are one of the more promising vaccine vectors because they can be easily constructed and because live adenovirus vaccines have been administered orally to large numbers of military recruits without adverse reactions. In order to examine the efficacy of human adenoviruses as vaccines we have studied the immunity induced by a recombinant adenovirus vector, AdgB2, which induces high level expression of herpes simplex virus (HSV) glycoprotein B (gB) in human and murine cells. Mice inoculated with AdgB2 produced antibodies specific for gB which neutralized HSV in the presence of complement. Although mice inoculated with AdgB2 showed no ill-effects after AdgB2 inoculation and we were unable to detect replication of human adenoviruses in mice, the mice were protected from a lethal challenge with HSV after a single inoculation with AdgB2. 相似文献
8.
Xu XG Chiou MT Zhang YM Tong DW Hu JH Zhang MT Liu HJ 《Journal of virological methods》2008,153(2):149-155
Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E(rns) is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E(rns) expressing histidine-tagged E(rns) with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E(rns) was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western-blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E(rns) glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E(rns) elicited significantly higher E(rns) antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E(rns) vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E(rns)-pseudotyped baculovirus as a CSFV vaccine. 相似文献
9.
Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. In the European Union the virus has been eradicated from the domestic pig population and prophylactic immunization has been banned. Nevertheless, intervention immunizations using marker vaccines are one possibility to deal with reintroduced CSFV. At present, baculovirus-expressed E2 protein is used as such a marker vaccine. However, this vaccine cannot fully protect against viral spread; hence the use of another subunit, or of a combination of two or more subunits, might be beneficial. Therefore the immunological effects of nonstructural protein 3 (NS3) on the humoral as well as the cellular arms of the immune system were investigated. Although effectors of both sides of the immune system were stimulated by application of recombinant NS3 protein, no protection against lethal CSFV challenge could be achieved. 相似文献
10.
Mutations in the carboxyl terminal region of E2 glycoprotein of classical swine fever virus are responsible for viral attenuation in swine 总被引:4,自引:0,他引:4
Risatti GR Holinka LG Fernandez Sainz I Carrillo C Kutish GF Lu Z Zhu J Rock DL Borca MV 《Virology》2007,364(2):371-382
We have previously reported [Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L. 2005a. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J. Virol. 79, 3787-3796] that chimeric virus 319.1v containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background of highly virulent CSFV strain Brescia (BICv) was markedly attenuated in pigs. To identify the amino acids mediating 319.1v attenuation a series of chimeric viruses containing CS E2 residues in the context of the Brescia strain were constructed. Chimera 357v, containing CS E2 residues 691 to 881 of CSFV polyprotein was virulent, while chimera 358v, containing CS E2 residues 882 to 1064, differing in thirteen amino acids from BICv, was attenuated in swine. Single or double substitutions of those amino acids in BICv E2 to CS E2 residues did not affect virulence. Groups of amino acids were then substituted in BICv E2 to CS E2 residues. Mutant 32v, with six substitutions between residues 975 and 1059, and mutant 33v, with six substitutions between 955 and 994, induced disease indistinguishable from BICv. Mutant 31v, with seven substitutions between residues 882 and 958, induced a delayed onset of lethal disease. Amino acids abrogating BICv virulence were then determined by progressively introducing six CS residues into 31v. Mutant 39v, containing nine residue substitutions, was virulent. Mutant 40v, containing ten residue substitutions, induced mild disease. Mutant 42v, containing twelve substitutions, and mutant 43v, with an amino acid composition identical to 358v, were attenuated in swine indicating that all substitutions were necessary for attenuation of the highly virulent strain Brescia. Importantly, 358v protected swine from challenge with virulent BICv at 3 and 28 days post-infection. 相似文献
11.
Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, 753RYLASLHKKALPTSV767, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues 753RYLASLHKKALPTSV767 on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues 761K, 763L and 764P were critical for the reactivity with mAbs. In addition, the up- and downstream residues 753R, 754Y, 755L and 765T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif 753RYLASLHKKALPT765, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition. 相似文献
12.
Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene 总被引:6,自引:0,他引:6
J. M. Hammond E. S. Jansen C. J. Morrissy M. M. Williamson A. L. M. Hodgson M. A. Johnson 《Archives of virology》2001,146(9):1787-1793
Summary. A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge. Accepted May 29, 2001 Received April 11, 2001 相似文献
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15.
Summary. Rinderpest virus haemagglutinin (H) protein was expressed in eukaryotic cells and the plasmid encoding H gene was used for
immunization of rabbits. The immunized rabbits were completely protected from lethal challenge with virulent lapinized rinderpest
virus. Similar results were also observed in rabbits vaccinated with tissue culture rinderpest (TCRP) vaccine. About 80% of
control rabbits vaccinated with empty vector (mock-vaccinated) died after challenge. The thermal reactions and clinical symptoms
were less intense in H-DNA vaccinated rabbits as compared to the other two groups. Marked lymphopenia was observed in TCRP
and mock-vaccinated rabbits after challenge, however, the H-DNA vaccinated rabbits showed lymphocytosis following challenge.
The ap-pearance of lymphocytosis in H-DNA vaccinated rabbits is an interesting finding which needs further investigation.
Received June 27, 2001 Accepted November 8, 2001 相似文献
16.
D Meyer A Aebischer M Müller B Grummer I Greiser-Wilke V Moennig MA Hofmann 《Virology》2012,433(1):45-54
The Erns glycoprotein of classical swine fever virus (CSFV) has been studied in detail concerning biochemical and functional properties, whereas less is known about its antigenic structure.In order to define epitopes recognized by CSFV-specific antibodies, the binding sites of seven Erns-specific monoclonal antibodies were investigated. Mapping experiments using chimeric Erns proteins, site-directed mutagenesis and an overlapping peptide library identified one antigenic region located between amino acids (aa) 55 to 110 on the Erns protein of CSFV Alfort/187. The domain comprises three linear motifs ?64TNYTCCKLQ72, 73RHEWNKHGW81, and 88DPWIQLMNR96, respectively, and two aa at position 102 and 107 that are crucial for the interaction with antibodies. Additionally, the presentation of the epitope in a correct conformation is mandatory for an efficient antibody binding. These findings allow a better understanding of the organization and the structure of the Erns and provide valuable information with regard to the development of Erns-based diagnostic tests. 相似文献
17.
Parchariyanon S Inui K Pinyochon W Damrongwatanapokin S Takahashi E 《Journal of virological methods》2000,87(1-2):145-149
A method for genetic grouping of classical swine fever viruses (CSFV) was developed based on the restriction fragment length polymorphism (RFLP) revealed by AvaII, BanII and PvuII digestion of RT-PCR amplified segments of the E2 gene. From inspection of the genetic sequences of Thai isolates and reference strains, the RFLP method was designed to be capable of differentiating all known genogroups and subgenogroups suggested by phylogenetic analysis of the CSFV E2 gene. The method was applied to 60 CSFV samples which included three genogroups and seven subgenogroups. Unlike previously described RFLP methods, the agarose gel patterns obtained from these samples were completely in agreement with the predicted RFLP patterns and enabled accurate genetic grouping of CSFV at the subgenogroup level. The simplicity of this method allows rapid CSFV genogrouping at diagnostic laboratories without sequencing facilities and provides a useful method for diagnosis as well as epidemiological investigation and control of classical swine fever outbreaks. 相似文献
18.
Sérgio Oliveira De Paula Danielle Malta Lima Rafael Freitas de Oliveira França Alessandra Cristina Gomes-Ruiz Benedito Antônio Lopes da Fonseca 《Archives of virology》2008,153(12):2215-2223
In an effort to develop a suitable DNA vaccine candidate for dengue, using dengue-3 virus (DENV-3) as a prototype, the genes
coding for premembrane (prM) and envelope proteins (E) were inserted into an expression plasmid. After selecting recombinant
clones containing prM/E genes, protein expression in the cell monolayer was detected by indirect immunofluorescence and immunoprecipitation
assays. After selecting three vaccine candidates (pVAC1DEN3, pVAC2DEN3 and pVAC3DEN3), they were analyzed in vivo to determine
their ability to induce a DENV-3-specific immune response. After three immunizations, the spleens of the immunized animals
were isolated, and the cells were cultivated to measure cytokine levels by ELISA and used for lymphoproliferation assays.
All of the animals inoculated with the recombinant clones induced neutralizing antibodies against DENV-3 and produced a T
cell proliferation response after specific stimuli. Immunized and control mice were challenged with a lethal dose of DENV-3
and observed in order to assess their survival capability. The groups that presented the best survival rate after the challenge
were the animals vaccinated with the pVAC3DEN3 clones, with an 80% survival rate. Thus, these data show that we have manufactured
a vaccine candidate for DENV-3 that is able to induce a specific immune response and protects mice against a lethal challenge. 相似文献
19.
A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses. 相似文献
20.
Myunghwan Jung Yun Ji Shin Ju Kim Seung-Bin Cha Won-Jung Lee Min-Kyoung Shin Seung Won Shin Moon-Sik Yang Yong-Suk Jang Tae-Ho Kwon Han Sang Yoo 《Archives of virology》2014,159(12):3219-3230
Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs. Therefore, two kinds of new candidates for oral vaccination have been developed based on the translation of the E2 gene of the SW03 strain, which was isolated from an outbreak of CSF in 2002 in Korea, in transgenic rice calli (TRCs) from Oriza sativa L. cv. Dongjin to express a recombinant E2 protein (rE2-TRCs). The expression of the recombinant E2 protein (rE2) in rE2-TRCs was confirmed using Northern blot, SDS-PAGE, and Western blot analysis. Immune responses to the rE2-TRC in mice and pigs were investigated after oral administration. The administration of rE2-TRCs increased E2-specific antibodies titers and antibody-secreting cells when compared to animals receiving the vector alone (p < 0.05 and p < 0.01). In addition, mice receiving rE2-TRCs had a higher level of CD8+ lymphocytes and Th1 cytokine immune responses to purified rE2 (prE2) in vitro than the controls (p < 0.05 and p < 0.01). Pigs receiving rE2-TRCs also showed an increase in IL-8, CCL2, and the CD8+ subpopulation in response to stimulation with prE2. These results suggest that oral administration of rE2-TRCs can induce E2-specific immune responses. 相似文献