结核杆菌Ag85A和mGM-CSF共同表达载体的构建与CTL活性的诱导

构建、鉴定结核杆菌Ag85A和细胞因子GM CSF共同表达质粒 ,为研究新型抗结核杆菌DNA疫苗提供新的策略。先从质粒pBSby5中扩增出结核杆菌Ag85A基因序列并插入到质粒pIRES上成为pI85A ,用PCR方法从pc mGM CSF质粒中扩增出mGM CSF ,构建于pI85A质粒上 ,成为共同表达质粒pI85AGM。然后转染 772 1细胞 ,进行蛋白的表达和鉴定。结果经酶切鉴定和DNA测序证实重组质粒构建正确。通过RT PCR、SDS PAGE、Westernblot检测证实Ag85A与mGM CSF基因的转录和蛋白表达正确。Ag85A与mGM CSF免疫组小鼠的CTL活性明显增强。表明细胞因子GM CSF和结核杆菌Ag85A共同表达载体构建成功 ,并能诱导小鼠的CTL活性 ,为研制新型结核病疫苗奠定了基础     

Comparison of Immune Responses Induced in Mice by Vaccination with DNA Vaccine Constructs Expressing Mycobacterial Antigen 85A and Interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.     

Comparison of immune responses induced in mice by vaccination with DNA vaccine constructs expressing mycobacterial antigen 85A and interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.     

探讨BCG初次免疫结核杆菌DNA疫苗加强免疫方案的效应

目的:探讨BCG初次免疫(BCG-prime),结核杆菌共表达DNA疫苗加强免疫(DNA疫苗-boost)的策略对小鼠的免疫效果。方法:将BCG及结核杆菌重组DNA疫苗依次免疫小鼠,通过检测CTL和NK细胞的杀伤活性和特异性淋巴细胞增殖,以及小鼠血清抗体及细胞因子的水平,观测BCG-prime、共表达结核杆菌Ag85A/GM-CSFDNA疫苗boost策略对小鼠的免疫效果。结果:采用prime-boost免疫策略组的小鼠CTL的杀伤活性明显增强、特异性淋巴细胞明显增殖、IFN-γ的水平明显增高,NK细胞杀伤活性与对照组相比也有一定提高,但未超过BCG单独免疫效果。免疫小鼠血清特异性抗体的滴度超过单独DNA疫苗免疫组。结论:在采用BCG-prime-结核杆菌DNA疫苗boost免疫策略后,能增强对小鼠的免疫效应,尤其是Th1型细胞免疫反应增强明显,为进一步在动物体内进行保护性效应试验的研究提供了实验依据。     

结核分枝杆菌Ag85B-MPT64融合基因疫苗的免疫效果观察

目的:研究并比较结核分枝杆菌(H37Rv)Ag85B、MPT64DNA.以及两者的融合基因(AM)的免疫原性。方法:雌性C57BL/6小鼠25只,随机分为5组,即A组(PBS)、B组(peDNA3.1)、C组(pcDNA/Ag85B)、D组(pcDNA/MPT64)和E组(pcDNA/AM)。分别于胫前肌注射7.5 g/L利多卡因和质粒混合物(1:4,100μL,含质粒70μg/次),间隔2 wk免疫 1次,共3次。末次免疫后4 wk取血分离血清测定总IgG,同时分离脾淋巴细胞,用PPD刺激后分别做脾淋巴细胞增殖实验(MTT比色法)和测定脾淋巴细胞培养上清中IFN-γ的水平。结果:Ag85B、MPT64和AM质粒DNA,均能诱导小鼠产生较高水平的PPD特异性IgG。免疫小鼠脾淋巴细胞体外经PPD刺激后,能产生特异性淋巴细胞增殖和分泌IFN-γ。peDNA/AM组IFN-γ的分泌水平明显高于pcDNA/Ag85B和pcDNA/MPT64免疫组(P<0.05)。结论:结核分枝杆菌Ag85B-MPT64融合基因疫苗,能在小鼠体内诱导特异性细胞和体液免疫。     

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.     

Enhanced cellular immune response elicited by a DNA vaccine fused with Ub against Mycobacterium tuberculosis

This study evaluated the immune response elicited by a Ub-fused Ag85A DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding Ag85A protein, Ub-fused Ag85A DNA vaccine (UbGR-Ag85A) and negative DNA vaccines, respectively. Ag85A DNA vaccine immunization induced a Th(l)-polarized immune response. The production of Th(l)-type cytokine (IFN-γ) and proliferative T cell responses was enhanced significantly in mice immunized with UbGR-Ag85A fusion DNA vaccine, compared with non-fusion DNA vaccine. Moreover, this fusion DNA vaccine also resulted in an increased relative ratio of IgG(2a) to IgG(l) and the cytotoxicity of T cells. IFN-γ intracellular staining of splenocytes indicated that UbGR-Ag85A fusion DNA vaccine activated CD4(+) and CD8(+) T cells, particularly CD8(+) T cells. Thus, this study demonstrated that the UbGR-Ag85A fusion DNA vaccine inoculation could improve antigen-specific cellular immune responses, which is helpful for protection against TB infection.     

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A

The contribution of CD8+ and CD4+ T cell-mediated effector functions against Mycobacterium tuberculosis infection elicited by i.m. vaccination with plasmid DNA encoding the immunodominant Ag85A antigen of M. tuberculosis was studied. Ag85A DNA-vaccinated beta2-microglobulin gene-deficient (beta2m-/-) mice, which lack CD8+ T cells, produced Ag85-specific antibodies and Th1 type cytokines similar to wild-type mice. Although beta2m-/- mice were more susceptible to M. tuberculosis infection, following vaccination they efficiently controlled bacterial replication in spleen and lungs 4 weeks post-infection. In contrast, mice lacking CD4+ T cells were neither sensitized by the Ag85A DNA vaccine to produce Ag85-specific antibodies or Th1 type cytokines nor did they contain a M. tuberculosis challenge infection. In addition, Ag85A DNA-vaccinated IFN-gamma gene knockout mice produced Ag85-specific antibodies and IL-2 but died rapidly following a M. tuberculosis challenge infection. Collectively, these data support the view that IFN-gamma-producing CD4+ T cells, independently of CD8+ T cells, may mediate the protective effect of the Ag85A DNA vaccine.     

Treatment of multi-drug-resistant tuberculosis in mice with DNA vaccines alone or in combination with chemotherapeutic drugs

The problems of tuberculosis (TB) and its drug resistances are very severe in China. New therapeutic agents or regimens to treat multi-drug-resistant tuberculosis (MDR-TB) are urgently needed. We studied the effects of Ag85A DNA vaccine alone or in combination with rifampin (RFP) or pyrazinamide (PZA) for the treatment of MDR-TB in mice. Ag85A DNA vaccine significantly increased the production of IFN-γ, but lowered the production of IL-4. Seventy female BALB/c mice infected with Mycobacterium tuberculosis clinical isolate HB361, which was resistant to RFP and isoniazid but sensitive to PZA, were treated with plasmid pVAX1, RFP, PZA, M. vaccae vaccine, Ag85A DNA, Ag85A DNA combined with RFP or PZA, respectively. Ag85A DNA vaccine alone or in combination with RFP or PZA reduced the pulmonary and splenic bacterial loads by 1.03-1.38 logs, respectively. Ag85A DNA combined with conventional chemotherapy for the treatment of MDR-TB might result in cure of MDR-TB in developing countries.     

结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。     

T-bet佐剂对Ag85B抗结核DNA疫苗的免疫调节

目的:构建以Th1转录因子T-bet为基因佐剂的Ag85B新型DNA疫苗,并研究其免疫调控作用。方法:RT-PCR法扩增出Ag85B基因和T-bet基因,克隆入pcDNA3.1质粒构建T-bet和Ag85B真核表达质粒,脂质体法转染重组质粒至RAW264.7细胞系,Western blot法检测质粒蛋白表达情况。3次肌肉注射免疫BALB/c小鼠,末次免疫2周后,ELISA法检测血清中抗Ag85B抗体滴度。同时将脾脏淋巴细胞悬液于Ag85B刺激下培养,ELISA法检测培养液中细胞因子分泌情况。结果:质粒蛋白成功表达,并且质粒剂量与质粒蛋白表达水平呈正相关。此外,T-bet/Ag85B不仅诱导IgG2a滴度显著增高伴随IgG1显著降低,而且还刺激IL-2/IFN-γ(Th1类)分泌增加伴随IL-4/IL-10(Th2类)减少。结论:T-bet增强抗Ag85B特异性IgG2a抗体反应,并诱导显著的Th1细胞优势免疫。     

Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon

There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-gamma)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2(d)) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2(b)) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-gamma.     

Tuberculosis DNA vaccine encoding Ag85A is immunogenic and protective when administered by intramuscular needle injection but not by epidermal gene gun bombardment

Immunogenicity and protective efficacy of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis were compared in BALB/c and C57BL (B6 and B10) mice immunized by intramuscular (i.m.) needle injection or epidermal gene gun (gg) bombardment. In BALB/c mice, gg immunization could induce elevated antibody and cytotoxic T lymphocyte responses with plasmid doses 50-fold lower than those required for i.m. immunization. Interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion, however, was much lower in gg-immunized than in i.m.-immunized BALB/c mice. On the other hand, C57BL mice reacted only very weakly to gg immunization, whereas elevated Ag85A-specific antibody, IL-2, and IFN-gamma responses (significantly higher than in BALB/c mice) were detected following vaccination by the i.m. route. Antibody isotypes were indicative of Th2 activation following gg injection of BALB/c and of Th1 activation following i.m. injection of C57BL mice. Finally, C57BL but not BALB/c mice were protected by i.m. Ag85A DNA immunization against intravenous M. tuberculosis challenge, as measured by reduced numbers of CFU in spleen and lungs, compared to animals vaccinated with control DNA. Gene gun immunization was not effective in either BALB/c or C57BL mice. These results indicate that i.m. DNA vaccination is the method of choice for the induction of protective Th1 type immune responses with the Ag85A tuberculosis DNA vaccine.     

Mucosal prime-boost vaccination for tuberculosis based on TLR triggering OprI lipoprotein from Pseudomonas aeruginosa fused to mycolyl-transferase Ag85A

Toll-like receptor (TLR) triggering is an important step in the induction of T helper (Th) type 1 T cells which are key players in protection against the intracellular pathogen Mycobacterium (M.) tuberculosis. Here we report on the construction of a fusion protein consisting of a tuberculosis vaccine candidate mycolyl-transferase antigen 85A (Ag85A, Rv3804c) coupled to the outer membrane lipoprotein I (OprI) from Pseudomonas (P.) aeruginosa, a documented TLR2/TLR4 trigger. Subcutaneous boosting with this fusion protein in the absence of adjuvant increased significantly the Ag85A-specific humoral but not cellular immune responses of Ag85A-DNA vaccinated mice. Intranasal priming of C57BL/6 mice with live, attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine, followed by intranasal boosting with OprI-Ag85A increased systemic and local antigen-specific interferon (IFN)-gamma and interleukin (IL)-2 responses in spleen, draining cervical and mediastinal lymph nodes and particularly in lung tissue, as compared to responses in mice only vaccinated with BCG vaccine. Despite enhanced immune responses, boosting with OprI-Ag85A did not increase protective efficacy against M. tuberculosis of either plasmid DNA or BCG vaccine in this experimental setting.     

Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A

The immunogenicity of a plasmid DNA vaccine incorporating Sindbis virus RNA replicase functions (pSINCP) and expressing antigen 85A (Ag85A) from Mycobacterium tuberculosis was compared with a conventional plasmid DNA vector encoding Ag85A. pSINCP-85A was highly immunogenic in mice and gave enhanced long-term protection against M. tuberculosis compared with the conventional vector.     

Improved tuberculosis DNA vaccines by formulation in cationic lipids

Mice were vaccinated with plasmid DNA (pDNA) encoding antigen 85A (Ag85A), Ag85B, or PstS-3 from Mycobacterium tuberculosis either in saline or formulated for intramuscular injections in VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine) (Vaxfectin; Vical, Inc., San Diego, Calif.) or for intranasal instillations in GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine). These two novel cationic and neutral colipid formulations were previously reported to be effective adjuvants for pDNA-induced antibody responses. The levels of Ag85-specific total immunoglobulin G (IgG) and IgG isotypes were all increased 3- to 10-fold by formulation of pDNA in Vaxfectin. The level of production of splenic T-cell-derived Th1-type cytokines (interleukin-2 and gamma interferon) in response to purified Ag85 and to synthetic peptides spanning the entire Ag85A protein was also significantly higher in animals vaccinated with pDNA formulated in Vaxfectin. Cytolytic T-lymphocyte responses generated by pDNA encoding phosphate-binding protein PstS-3 in Vaxfectin were better sustained over time than were those generated by PstS-3 DNA in saline. Intranasal immunization with Ag85A DNA in saline was completely ineffective, whereas administration in GAP-DLRIE:DOPE induced a positive Th1-type cytokine response; however, the extent of the latter response was clearly lower than that obtained following intramuscular immunization with the same DNA dose. Combined intramuscular and intranasal administrations in cationic lipids resulted in stronger immune responses in the spleen and, more importantly, in the lungs as well. Finally, formulation in Vaxfectin increased the protective efficacy of the Ag85B DNA vaccine, as measured by reduced relative light unit counts and CFU counts in the spleen and lungs from mice challenged with bioluminescent M. tuberculosis H37Rv. These results may be of importance for future clinical use of DNA vaccines in humans.     

结核分支杆菌Ag85A DNA疫苗免疫治疗作用的研究

目的:研究结核分支杆菌Ag85A DNA疫苗的免疫治疗作用。方法:用结核分支杆菌H37Rv腹腔内注射BALB/c小鼠 2个月后,将小鼠随机分为 5组,分别用生理盐水(A)、载体质粒(B)、卡介苗(C)、维卡菌苗(D)和 Ag85A DNA疫苗(E)免疫小鼠;用ELISA法检测血清抗体;免疫治疗2月和5月时,分别取肺、肝和脾脏观察病理改变、称取重量、作菌落计数、肺组织涂片及脾淋巴细胞转化试验。结果:DNA疫苗组抗原特异性抗体不同程度升高。免疫治疗2月时,各组鼠脾淋巴细胞转化率无显著差异;各治疗组肺组织涂片细菌数和肺菌落计数均比对照组不同程度地减少;C和B组的肺病变程度较轻。免疫治疗5月时,各治疗组鼠脾淋巴细胞转化率均显著增高;各组肺、脾细菌数无显著差别比和D组的肺未见明显病变。结论:DNA疫苗似乎具有一定的免疫治疗效果。     

结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性

目的通过原核表达获得结核分枝杆菌Ag85A蛋白。方法用PCR从结核分枝杆菌H37Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达。用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性。利用PCR技术鉴定fbpA基因在不同分枝杆菌的分布。结果32 ku的Ag85A蛋白获得高效表达和纯化。表达Ag85A蛋白的fbpA基因在结核分枝杆菌H37Rv、H37Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达。结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高。结论重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性。     

Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant

Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.     

口服Ag85A DNA疫苗表达产物在脾脏的分布

目的 检测口服Ag85A DNA疫苗表达产物在脾脏内的分布,为阐明口服DNA疫苗可诱导全身性免疫应答的机制提供依据。方法 将本实验室构建的pCDNA3．1^＋-Ag85A真核表达重组质粒转化感受态大肠杆菌DH5α进行扩增,无内毒素抽提纯化,进一步用脂质体包裹制成口服重组Ag85A DNA疫苗。将C57BL／6小鼠随机分为2组,即生理盐水组和DNA疫苗组。分别将生理盐水和Ag85A DNA疫苗以灌胃方式投给各组小鼠,共免疫3次,每次间隔14d,末次免疫后14d处死小鼠,取脾,免疫组化、免疫荧光法检测Ag85A表达产物在脾脏的分布情况。结果 Ag85A重组DNA疫苗的表达产物在小鼠脾脏白髓、边缘区和红髓的脾索处有广泛分布,在边缘区及红髓的脾索处的检出强度高于白髓。免疫组化结果中边缘区与白髓比较t=3．039,P〈0．05;红髓的脾索与白髓比较t=3．068,P〈0．05;边缘区与红髓的脾索比较t=1．750,P〉0．05。免疫荧光结果中边缘区与白髓比较t=3．144,P〈0．05;红髓的脾索与白髓比较t=3．098,P〈0．05;边缘区与红髓的脾索比较t=1．369,P〉0．05。结论口服脂质体包裹的DNA疫苗的表达产物存在于脾脏,表明经口途径接种的DNA疫苗可能会在脾脏诱导全身性免疫应答的产生。