Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫诱导Th1和Th17细胞免疫应答抵抗肺炎链球菌感染 *

目的 探索更有效的肺炎链球菌DNA疫苗和疫苗免疫策略,并探究其中的保护机制。方法 构建重组质粒pcDNA3-dnaJ并表达DnaJ蛋白,实验分别设置重组质粒pcDNA3-dnaJ/蛋白DnaJ免疫小鼠组及单独质粒pcDNA3-dnaJ免疫小鼠组,分别比较肺炎链球菌菌株攻毒后小鼠鼻腔灌洗液细菌载量及生存率,采用ELISA检测免疫小鼠血清抗体效价及炎症因子,流式细胞术分析体外BMDCs激活情况及Th1和Th17细胞免疫应答。结果 质粒pcDNA3-dnaJ免疫3次可诱导血清中抗原特异性抗体的产生,并减少肺炎链球菌攻毒后鼻咽部的细菌载量,但在防止致死性感染方面效果较差。然而,与重复质粒DNA接种三次相比,pcDNA3-dnaJ 1次/ DnaJ蛋白加强1次的免疫策略可以显著减少鼻咽中的肺炎链球菌定植,并能够更好的预防致死性感染。此外,与DNA质粒加强免疫相比,DnaJ蛋白加强免疫后可产生更高水平的IFN-γ和IL-17A。结论 重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫可能通过活化树突状细胞,进而诱导Th1和Th17细胞免疫应答,抵抗肺炎链球菌感染。     

日本血吸虫新抗原基因的筛选、克隆、 表达和免疫效果研究

为了寻找日本血吸虫 (Schistosoma japonicum, Sj) 新的疫苗候选基因并进行免疫效果研究,用 Sj 雌虫抗原免疫家兔制备血清,对Sj成虫 cDNA 文库进行免疫筛选,将获得的新基因 ( 命名为Sj-F1, GenBank 登录号为 AY261995) 克隆入原核表达载体 pTWIN1 和真核表达载体 pcDNA3 ,经 PCR 、限制性酶切筛选和鉴定阳性重组子. 将 pTWIN1/Sj-F1 质粒转化大肠杆菌 ER2566,在低温和低 IPTG 浓度下诱导表达可溶性重组融合蛋白 (rSj-F1/intein2),并经 SDS- 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和蛋白质印迹 (Western blot) 分析鉴定. 将 pcDNA3/Sj-F1 质粒转化大肠杆菌 ER2502 ,大量制备 DNA 疫苗. 用重组融合蛋白和 DNA 疫苗免疫小鼠,末次免疫后 2 周用Sj尾蚴进行攻击感染. 感染后 42 天剖杀冲虫,计算减虫率和减卵率. 感染前采血用 ELISA 法检测抗体. 免疫保护效果测定显示：重组蛋白疫苗以 FCA 作佐剂经皮下免疫和以壳聚糖作佐剂经粘膜免疫分别获得了 28.07%、 24.69% 的减虫率和 48.30% 、 46.38% 的减卵率; DNA 疫苗 (pcDNA3/Sj-F1) 单独免疫获得了 18.47% 的减虫率和 35.06% 的减卵率;用 DNA 疫苗启动免疫后用重组蛋白疫苗经皮下加强免疫,减虫率和减卵率分别提高到了 40.42% 和 56.17%;用 DNA 疫苗启动免疫后用重组蛋白疫苗经黏膜加强免疫,减虫率和减卵率增高更明显,分别提高到了 42.38% 和 62.87%. 结果表明,Sj-F1 重组蛋白疫苗及 DNA 疫苗均可诱导小鼠产生部分抗血吸虫感染的保护力,两者联合免疫保护效果优于单一疫苗.     

MAGE-3 DNA疫苗的构建及其免疫效果的观察

通过RT PCR方法扩增MAGE 3cDNA ,以pcDNA3 1+为载体 ,构建重组表达质粒pcDNA3 1 MAGE 3。重组质粒用脂质体转染鼠B16细胞 ,经RT PCR、细胞免疫染色及免疫印迹法鉴定转化细胞中MAGE 3的表达。以 10 0 μg质粒剂量肌肉注射接种小鼠 ,间隔 10天 ,共 3次 ,以空载体和PBS为对照。结果 ,重组质粒免疫的小鼠 ,其脾淋巴细胞对MAGE 3阳性靶细胞的杀伤活性为 51 0 8± 7 41% ,与空载体组 (8 44± 1 89% )及PBS组 (5 76± 1 75% )相比 ,差异有显著性 (P <0 0 1) ,而对MAGE 3阴性靶细胞的杀伤活性分别为 8 2 1± 1 65% ,7 68± 1 56%和 5 13±1 42 % ,其差异无显著性 ;MAGE 3DNA疫苗组免疫血清 1∶15稀释时能检测到抗MAGE 3抗体 ,脾细胞培养上清中Th1类细胞因子IFN γ、IL 2水平明显升高 ,外周血中CD4+、CD8+T细胞也提高 ,小鼠肿瘤的生长速度明显减慢 ,与对照组相比 ,差异显著 (P <0 0 1)。说明MAGE 3重组质粒免疫小鼠可以诱导小鼠产生特异性的体液和细胞免疫应答     

The ‘Co-Delivery’ Approach to Liposomal Vaccines: Application to the Development of influenza-A and hepatitis-B Vaccine Candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination ‘co-delivery’ and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell – a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

The 'co-delivery' approach to liposomal vaccines: application to the development of influenza-A and hepatitis-B vaccine candidates

DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination 'co-delivery' and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell--a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.     

DNA immunization with a plasmid carrying the gene of hepatitis C virus protein 5A (NS5A) induces an effective cellular immune response

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti- NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against a broad spectrum of NS5A epitopes as assessed by T-cell proliferation and secretion of antiviral cytokines IFN-γ and IL-2. In T-cell stimulation in vitro experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.     

Immunogenicity and Efficacy of Single Antigen Gp63, Polytope and PolytopeHSP70 DNA Vaccines against Visceral Leishmaniasis in Experimental Mouse Model

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.     

Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice

To analyze the immune responses of DNA vaccine encoded different gene fragments of severe acute respiratory syndrome coronavirus (SARS-Cov), SARS-Cov gene fragments of membrane (M), nucleocapsid (N), spike a (Sa), and spike b (Sb) proteins were cloned into pcDNA3.1 (Invitrogen) vector to form plasmids pcDNAM, pcDNAN, pcDNASa, and pcDNASb, respectively. After mice were immunized intramuscularly with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmid, blood was collected and serum was separated. Humoral immune response was detected with the enzyme-linked immunosorbent assay, and cellular immune response of SARS-Cov DNA vaccines was detected with lymphoproliferation assay and cytotoxic T lymphocyte assay. Results show that cellular and humoral immune responses can be detected after immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmids in BALB/c mice. However, pcDNAM stimulated the highest cellular immune response than other plasmids, and pcDNASa-pcDNASb stimulated the highest humoral immune response in week 12. The present results not only suggest that DNA immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also to illustrate that gene immunization with these SARS DNA vaccines different immune response characters.     

Dissecting the Immune Response to Moloney Murine Sarcoma/Leukemia Virus-Induced Tumors by Means of a DNA Vaccination Approach

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag_85–93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env_189–196, SSWDFITV), originally described in the B6.CH-2^bm13 mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.     

Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein

To evaluate the peptidoglycan-associated lipoprotein (PAL) antigen of Legionella pneumophila as a vaccine candidate, mice were immunized intramuscularly with pcDNA3-PAL and intraperitoneally with recombinant PAL (t-rPAL), which were compared for their ability to induce PAL-specific immune responses. The t-rPAL protein induced PAL-specific IgG antibody production significantly more than did pcDNA3-PAL. The IgG2a and IgG1 production was predominant after pcDNA3-PAL and t-rPAL administration, respectively. In particular, pcDNA3-PAL induced much higher PAL-specific cytotoxic T-lymphocyte responses than did t-rPAL. Furthermore, in vivo, CD19+ B-cell populations were dramatically increased by t-rPAL vaccination, suggesting a B-cell immunomodulatory activity of the lipoprotein. The PAL antigen was also conserved among Legionella species, as determined by PCR and immunoblot analyses. These results support a potential use of the t-rPAL protein and in particular DNA vaccines against Legionella infections.     

The Effect of Bovine IFN-α on the Immune Response in Guinea Pigs Vaccinatedwith DNA Vaccine of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) belongs to thegenus Aphthovirus of the family Picornavidae. The FMDVgenome is a copy of positive-sense, single-stranded RNA,which contains one large open reading frame (ORF). TheORF is translated into a polypeptide, which undergoesautoproteolytic cleavage to produce the structural and non-structural proteins and ultimately forms mature viral pro-teins [1,2]. FMD is caused by the FMDV, which is a highly conta-gious vesicular disease of cloven-hoofe…     

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.     

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.     

Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein,a candidate for DNA vaccine

Background:

DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed.

Methods:

To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed.

Results:

The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR.

Conclusion:

Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.Key Words: Brucella melitensis, omp31, DNA Vaccine, pcDNA3.1     

Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge

Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats.     

Asia Ⅰ型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位.本研究设计和合成了由Asia Ⅰ型FMDV VP1蛋白136～160aa和198～211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因.利用BamH I、EcoR I和Xho I位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG.100μg FshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200 ID_(50)剂量FMDV攻击时得到了完全保护.由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防.     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamydia-induced upper genital tract gross pathology and histopathological characterization were also detected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were significantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vaccinated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against pathological consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immunization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.