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1.
Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.  相似文献   

2.
Induction of effective immunity requires the delivery of a protective antigen with appropriate co-stimulatory signals. For bovine viral diarrhoea virus (BVDV) this antigen is the major viral glycoprotein E2. Neutralising antibodies are directed towards the E2 protein and passive transfer of antibodies in serum or colostrum can completely protect against viral infection. DNA vaccination of mice with a construct encoding the E2 glycoprotein induced neutralising antibody levels that were potentially sufficient to prevent virus replication in a challenge system. The co-delivery of interleukin-2 (IL-2) further enhanced the levels of antibody raised. The strong IgG2a component of the antigen-specific antibody suggests a Th1 bias to the immune response induced following vaccination.  相似文献   

3.
A DNA vaccine expressing glycoprotein C (gC) of bovine herpesvirus-1 (BHV-1) was evaluated for inducing immunity in bovines. The plasmid encoding gC of BHV-1 was injected six times intramuscularly or intradermally into calves at monthly intervals. After immunization by both routes neutralizing antibody and lymphoproliferative responses developed. The responses in the intradermally immunized calves were better than those in calves immunized intramuscularly. However, the intradermal (i.d.) route was found to be less efficacious when protection against BHV-1 challenge was compared. Following intranasal BHV-1 challenge, all immunized calves demonstrated a rise in IgG antibody titre on day 3, indicating an anamnestic response. The control non-immunized calf developed a neutralizing antibody response on day 7 post-challenge. The immunized calves showed a slight rise in temperature and mild clinical symptoms after challenge. The intramuscularly immunized calves showed earlier clearance of challenge virus compared with intradermally immunized calves. These results indicate that DNA immunization with gC could induce neutralizing antibody and lymphoproliferative responses with BHV-1 responsive memory B cells in bovines. However, the immunity developed was not sufficient to protect calves completely from BHV-1 challenge.  相似文献   

4.
Bovine herpesvirus 1 (BoHV-1) has frequently been used as a model for testing parameters affecting DNA immunisation in large animals like cattle. However, the selection of target antigens has been poorly studied, and most of the experiments have been conducted in mice. In the present study, we demonstrated in cattle that a DNA vaccine encoding BoHV-1 glycoprotein gD induces higher neutralising antibody titres than vaccines encoding BoHV-1 gC. Additionally, we show that a DNA vaccine encoding a secreted form of gD induces a higher immune response than a vaccine encoding full-length gD. However, the enhanced immunogenicity associated with the secretion of gD could not be extended to the glycoprotein gC. The current study also describes for the first time the development and the evaluation of a DNA vaccine encoding the major tegument protein VP8. This construct, which is the first BoHV-1 plasmid vaccine candidate that is not directed against a surface glycoprotein, induced a high BoHV-1 specific cellular immunity but no humoral immune response. The calves vaccinated with the constructs encoding full-length and truncated gD showed a non-significant tenfold reduction of virus excretion after challenge. Those calves also excreted virus for significantly (p < 0.05) shorter periods (1.5 days) than the non-vaccinated controls. The other constructs encoding gC and VP8 antigens induced no virological protection as compared to controls. Altogether the DNA vaccines induced weaker immunity and protection than conventional marker vaccines tested previously, confirming the difficulty to develop efficient DNA vaccines in large species.  相似文献   

5.
The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.  相似文献   

6.
We examined primary and memory isotype-specific antibody responses directed against pseudorabies virus in serum and mucosal fluids of pigs with and without passively acquired maternal antibody, and we studied the relationship between these responses and protection against virus challenge. Pigs were inoculated intranasally with the virulent NIA-3 strain or the avirulent Bartha strain, or they were inoculated IM with an inactivated vaccine containing the Phylaxia strain. Ten weeks later, all pigs were challenge-exposed intranasally with strain NIA-3. Only pigs that were without passively acquired antibody at the time they were inoculated with virulent virus appeared to have complete protective immunity against challenge exposure, as evidenced by lack of clinical signs of pseudorabies and lack of virus excretion. In contrast, pigs inoculated with strain Bartha or with the inactivated vaccine developed fever, had a period of growth arrest, and excreted virus after challenge exposure. In pigs without passively acquired antibody, intranasal inoculation with strains NIA-3 or Bartha was followed by primary IgM and IgA responses in serum and in oropharyngeal fluid as well as primary IgG1 and IgG2 responses in serum. Intramuscular inoculation with the inactivated vaccine induced primary serum IgM, IgG1, and IgG2 responses, but no mucosal responses. Challenge exposure of pigs that had been inoculated with the Bartha strain or the inactivated vaccine was followed by clear memory responses in serum and in oropharyngeal fluid. In contrast, challenge exposure of pigs that had been inoculated by the virulent NIA-3 strain was not followed by memory responses.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-gamma level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-gamma was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-gamma production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.  相似文献   

8.
应用PCR方法从包含伪狂犬病病毒（PRV）Fa株糖蛋白gD基因的重组质粒pB13中扩增出糖蛋白gD基因去信号肽片段，将其克隆到大肠埃希氏菌表达载体pThiohisA中。测序结果表明，糖蛋白gD基因去信号肽片段长1155bp，与PRVFa株糖蛋白gD基因完全一致。经1mmol／L IPTG诱导后，重组质粒pThiohisA-gD在大肠埃希氏 菌XL1-blue、Top10、DE3和DH5a中均得到表达，其中在宿主菌XL 1-blue中表达量最高。Western-blotting结果显示，大肠埃希氏菌XL1-blue表达的糖蛋白gD具有免疫原性。  相似文献   

9.
参照GenBank发表的猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位的编码区基因序列,设计一对引物,通过PCR扩增后,将约为600bp的目的片段克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32(a)的T7启动子下游,构建的重组质粒pET-gB经IPTG诱导,在大肠杆菌BL21(DE3)中获得了高效表达。SDS-PAGE结果显示,表达产物分子量约为42.4KDa,主要以包涵体形式存在。BandScan分析表明,表达量约占菌体蛋白的60.5%。利用His亲和层析方法得到了纯化的表达产物。Western blotting结果显示,重组蛋白能与阳性血清发生特异性反应,具有较好的抗原反应原性,可以作为检测用抗原。  相似文献   

10.
Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 microg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses.  相似文献   

11.
Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.  相似文献   

12.
This study was performed to determine whether electroporation can be used to enhance the efficacy of a DNA vaccine against pseudorabies virus (PrV) in pigs. Immune responses to PrV were measured in pigs following a single intramuscular injection of plasmids encoding PrV glycoprotein B, with or without electroporation. Plasmid injection coupled with electroporation increased production of specific antibodies against PrV and peripheral blood mononuclear cells proliferated in response to stimulation with PrV glycoproteins. These results show that electroporation can improve the performance of a DNA vaccine against PrV in pigs. However, additional work is required to maximise the effectiveness of the vaccination protocol.  相似文献   

13.
Foot-and-mouth disease (FMD) is one of the most devastating animal diseases, affecting all cloven-hoofed domestic and wild animal species. Previous studies from our group using DNA vaccines encoding FMD virus (FMDV) B and T cell epitopes targeted to antigen presenting cells, allowed demonstrating total protection from FMDV homologous challenge in those animals efficiently primed for both humoral and cellular specific responses (Borrego et al. Antivir Res 92:359-363, 2011). In this study, a new DNA vaccine prototype expected to induce stronger and cross-reactive immune responses against FMDV which was designed by making two main modifications: i) adding a new B-cell epitope from the O-serotype to the B and T-cell epitopes from the C-serotype and ii) using a dual promoter plasmid that allowed inserting a new cistron encoding the anti-apoptotic Bcl-xL gene under the control of the internal ribosomal entry site (IRES) of encephalomyocarditis virus aiming to increase and optimize the antigen presentation of the encoded FMDV epitopes after in vivo immunization. In vitro studies showed that Bcl-xL significantly prolonged the survival of DNA transfected cells (p?<?0.001). Accordingly, vaccination of Swiss out-bred mice with the dual promoter plasmid increased the total IgG responses induced against each of the FMDV epitopes however no significant differences observed between groups. The humoral immune response was polarized through IgG2a in all vaccination groups (p?<?0.05); except peptide T3A; in correspondence with the Th1-like response observed, a clear bias towards the induction of specific IFN-γ secreting CD4+ and CD8+ T cell responses was also observed, being significantly higher (p?<?0.05) in the group of mice immunized with the plasmid co-expressing Bcl-xL and the FMDV B and T cell epitopes.  相似文献   

14.
The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.  相似文献   

15.
Gene immunization can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. We constructed plasmid vectors expressing the full-length Vnukovo-32 rabies virus glycoprotein G under the control of CMV IE promoter and enhancer, adenovirus tripartite leader sequences and poly A signal of SV40. The gene vaccines were evaluated for the ability to elicit neutralizing antibodies and to protect BALB/c mice against lethal rabies virus challenge. First, mice were injected intramuscularly (i.m.) into the left hind leg and by the intradermoplantar (i.d.p.) route with equal amounts of plasmid DNA (0.25-0.1 mg). Two weeks later, immunization was boosted with an additional dose of the DNA. The immunized mice were challenged by intracerebral (i.c.) inoculation of CVS-27 (10-50 LD50) rabies virus. All mice produced anti-rabies virus neutralizing antibodies with a titre of > or = 1:45 after immunization with 0.1-0.4 mg of DNA. In challenge experiments, 83 to 91.6% protection was observed. These results confirm that a DNA vaccine could be a simple and effective solution for preventing the spread of rabies.  相似文献   

16.
Previously, we have reported that the injection of an expression vector containing Herpes simplex virus (HSV) Glycoprotein D-1 (gD-1) generated a significant antibody response in mice and protected them against HSV lethal challenge. We tested its potential to induce antibody and cell mediated immune responses in latently infected mice. Positive control group (KOS) and HSV gD-1 vaccinated mice demonstrated protection against a lethal ocularly challenge of 10(5.5) plaque-forming units (pfu)/eye of wild HSV-1 versus negative control groups. For neutralizing antibody titers, delayed-type hypersensitivity (DTH), lymphocyte proliferation responses, clinical evaluation and survival following lethal challenge, no considerable difference was observed between mice vaccinated with DNA plasmid and those vaccinated with KOS. KOS-vaccinated mice demonstrated the ability to completely prevent latency whereas DNA vaccinated group showed some degree of protection and displayed less latency than negative control groups and had considerably high levels of IFN-gamma and strong CTL responses versus negative control groups. It can be concluded that although immunization with the DNA vaccine is more effective in both protecting mice and induction of immune response, however it could not completely block the latent infection in sensory nerves.  相似文献   

17.
Recently we have demonstrated, with a DNA vaccine, that the immediate early protein (IE180) of pseudorabies virus provides a moderate level of protection in mice. In order to improve its immunogenicity and protective capacity, this IE180 DNA vaccine was delivered to C3H/HeJ mice either in combination with an IL-2 expressing plasmid or complexed with cationic liposomes. Co-delivery of the vaccine and IL-2 DNA by gene gun resulted in seroconversion in 5/5 of the vaccinated mice after a single administration, whereas two intramuscular (i.m.) injections were required to achieve seroconversion in all mice. Antibody and delayed-type hypersensitivity responses were augmented in mice, which received the DNA vaccine and the IL-2 gene compared to those of mice receiving the DNA vaccine alone. In addition, the time of death after challenge was significantly delayed in mice, which received the IL-2 gene. The proportion of surviving mice (40%), however, was similar to that obtained in mice which received the vaccine alone by gene gun. Liposome-mediated vaccine delivery also resulted in a higher rate of seroconversion when compared with that induced by the naked DNA vaccine. Thus, all vaccinated mice seroconverted after either two i.v. or three i.m. injections of the liposome/DNA complex, with 40 and 25% of these mice being protected against challenge, respectively. These data support that co-administration of the IE180 DNA vaccine with the IL-2 gene or delivery in liposomes are two effective approaches to increase its immunogenicity.  相似文献   

18.
Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulations. Here we assessed the potential for a combined vaccine based on plasmids encoding the membrane-anchored or secreted forms of bovine herpesvirus type 1 (BHV-1) glycoprotein B and D (gB and gD) to induce neutralizing and cell mediated immune responses in mice. Animals were injected by intramuscular, subcutaneous and intranasal routes. Mice immunized with the combined vaccine containing the secreted forms of BHV-1 glycoproteins developed higher titers of anti-BHV-1 neutralizing antibodies, compared to wild type gB/gD combined plasmids and to single plasmid injected groups. Cellular immunity was also developed in mice immunized with combined vaccines, whereas low or no response were observed in single plasmid injected animals. The data suggest the potential use of this combined vaccine in in vivo trials of calves, in order to evaluate its protective efficacy.  相似文献   

19.
Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscle has been shown to result in protective immunity against lethal infection. Nevertheless, such DNA vaccines are still less efficient than some attenuated or killed live vaccines. One way to increase DNA vaccine efficacy is to improve the vectorisation system at the molecular level, thereby enhancing the rate of in vivo-produced immunogen protein and consequently specific acquired immunity. The present study compared the effectiveness of the protein expression system depending on Sindbis virus (SIN) replicase [J. Virol. 70 (1996) 508] with that of more classical pcDNA3 plasmid. Pigs were vaccinated twice at 3-week interval with a mixture of three pcDNA3 plasmids expressing gB, gC and gD (designated as PRV-pcDNA3) or a mixture of three SIN plasmids expressing the same glycoproteins (PRV-pSINCP), and were challenged with a highly virulent PRV strain. The two DNA vaccines induced PRV-specific T cell-mediated immune response characterized by very low levels of IFN-gamma mRNA in PBMC after in vitro antigen-specific stimulation. Very low levels of neutralizing antibodies (NAb) were also obtained in sera following DNA injection(s). A second DNA injection did not boost immune responses. After a lethal challenge, high levels of IFN-gamma mRNA and high NAb response were induced in all DNA-vaccinated pigs, regardless of the vector used. Therefore, the two eukaryotic expression systems showed comparable efficacy in inducing antiviral immunity and clinical protection against PRV in pigs. This suggests that SIN DNA-based vector immunizing potential may differ according to antigen and/or host.  相似文献   

20.
根据伪狂犬病病毒闽Ａ株gE基因表位抗原编码区的序列与身份种真核表达载体pPICZaA、pAcGP67A序列与特性分别设计了两对ＰＣＲ引物。通过ＰＣＲ方法扩增到了两端具有不同酶切位点的gE基因表位抗原编码片段。将这２个片段分别克隆到pPICZaA与pAcGP67A载体，转化大肠杆菌ＴＯＰ１０菌档及ＸＬ１－Ｂlue菌株，获得了含伪狂犬病病毒闽Ａ株gE基因表位抗原编码区的重组质粒pICZaA-FS与pAcGP67A-FS。序测定结果显示两个重组质粒中插入片段的大小与方向均正确。  相似文献   

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