轮状病毒组特异性抗原VP6在重组腺病毒中的表达及其免疫学效果的研究

目的 构建可表达A组轮状病毒组特异性抗原VP6的非复制型重组腺病毒载体，并对其生物学和免疫学性质进行研究。方法 将人轮状病毒VP6基因插入腺病毒载体pShuttle-CMV中，通过细胞内同源重组方法获得重组腺病毒DNA，将其转染293细胞获得重组腺病毒。用聚合酶链反应（PCR）及Southern blot方法，检测目的基因在重组腺病毒中的整合，用Western blot检测VP6的表达。通过灌胃和滴鼻两种途径对小鼠进行免疫，并对免疫后小鼠体液和粘膜免疫进行分析。结果 得到了重组腺病毒rvAd-VP6,VP6基因整合于腺病毒基因且中，在293细胞中可稳定表达。2次免疫后灌胃和滴鼻两组小鼠均产生明显免疫应答，血清IgG抗体滴度分别为1：1000和1：10000－1：100000。除了血清IgG外，小鼠还产生了较强的针对轮状病毒的血清IgA，滴度为1：10－1：100。滴鼻组在肺灌洗液和肠匀浆液中均可检测到分泌型IgA(sIgA)，灌胃组仅在肠道检测到sIgA。滴鼻组的免疫学效果明显优于灌胃组。结论 人轮状病毒VP6基因重组腺病毒载体的成功构建及所取得的良好免疫学效果，为我国具有自主知识产权的新型轮状病毒基因工程疫苗的研制奠定了基础。     

滴鼻、口服及肌肉注射途径对轮状病毒VP4重组腺病毒免疫小鼠的体液免疫效果比较

目的：比较滴鼻、口服及肌肉注射免疫途径，对重组腺病毒刺激小鼠产生的体液免疫反应效果。方法：剂量为10^8PFu的表达轮状病毒VP4的重组腺病毒经滴鼻、口服、肌肉注射分别免疫BALB／c小鼠，相同免疫剂量、免疫途径加强免疫2次，采集小鼠的血清、粪便及小肠样本，用ELISA检测轮状病毒VP4特异的IgG和IgA。实验设立了腺病毒载体对照组和PBS对照组。结果：经3种途径免疫的重组腺病毒都刺激小鼠产生了轮状病毒VP4特异的血清和肠道抗体，与对照组相比，有统计学意义（JP〈0．05）。在免疫后的第7周，滴鼻组血清VP4特异LgG达到最高值（GMT=15000），在免疫后的第8周，肠道抗体达到最高值（GMT），其中IgG为74．8，IgA为36。结论：滴鼻免疫是3种免疫途径中最有效的。     

腺病毒载体表达轮状病毒G1型外壳蛋白VP7及其诱导的免疫应答研究

构建表达地方流行株轮状病毒G1型外壳蛋白VP7的复制缺陷型重组腺病毒,免疫小鼠评价其体液及细胞免疫反应效果,探讨基因工程轮状病毒疫苗的实验基础及可行性。RT-PCR扩增轮状病毒VP7基因并克隆至pshuttle-CMV穿梭质粒,pshuttle-VP7与腺病毒骨架质粒同源重组后转染293细胞包装重组腺病毒rAd-VP7。RT-PCR、western blot检测rAdVP7在体外细胞中的转录及表达。rAd-VP7经肌注或滴鼻免疫小鼠后检测其血清IgG、肠道IgA及中和抗体效价;流式、ELISPOT检测淋巴细胞亚群变化及IFN-γ分泌情况。结果显示ELISA可检测到免疫小鼠血清IgG和滴鼻组肠道IgA抗体的产生;肌注和滴鼻免疫组中和抗体平均滴度分别为228和322.5;免疫小鼠脾细胞IFN-γ的分泌增加。表达轮状病毒VP7的重组腺病毒不但能够激发体液及细胞免疫反应,滴鼻免疫途径还可诱导粘膜免疫应答。     

密码子优化增强轮状病毒VP6基因重组腺病毒的体液免疫和攻毒保护效果的研究

目的 以表达野生型RVVP6基因的重组病毒rvAdVP6为对照,在小鼠模型上观察基因优化重组腺病毒rvAdVP6(o)的体液免疫效果和攻毒保护效果.方法 6到8周雌性BALB/c小鼠随机分组,通过等量病毒(每次108TCID50/只)3次滴鼻免疫,检测3次免疫后的血清抗体;并用鼠轮状病毒进行攻毒实验,检测不同组的免疫保护效果.结果 三次免疫rvAdVP6(o)产生的抗VP6血清IgG抗体水平均明显高于rvAdVP6;鼠轮状病毒攻击后检测小鼠的排毒量,发现rvAdVP6(o)免疫组的RV排毒量明显少于rvAdVP6.结论 在等量重组腺病毒免疫的情况下,优化后的VP6重组腺病毒在体液免疫和攻毒保护方面,均优于优化前.     

密码子优化增强轮状病毒VP6基因重组腺病毒的体液免疫和攻毒保护效果的研究

目的以表达野生型RVVP6基因的重组病毒rvAdVP6为对照,在小鼠模型上观察基因优化重组腺病毒rvAdVP6（o）的体液免疫效果和攻毒保护效果。方法6到8周雌性BALB／c小鼠随机分组,通过等量病毒（每次108TCID50／只）3次滴鼻免疫,检测3次免疫后的血清抗体;并用鼠轮状病毒进行攻毒实验,检测不同组的免疫保护效果。结果三次免疫rvAdVP6（o）产生的抗VP6血清IgG抗体水平均明显高于rvAdVP6;鼠轮状病毒攻击后检测小鼠的排毒量,发现rvAdVP6（o）免疫组的RV排毒量明显少于rvAdVP6。结论在等量重组腺病毒免疫的情况下,优化后的VP6重组腺病毒在体液免疫和攻毒保护方面,均优于优化前。     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

鼠疫F1-V重组蛋白疫苗滴鼻免疫应答效果的研究

目的 以重组霍乱毒素B亚单位（rCT-B）为鼠疫F1-V重组蛋白的佐剂制备黏膜疫苗,观察小鼠诱导的黏膜免疫和系统免疫应答效果。方法以制备的鼠疫黏膜疫苗滴鼻免疫小鼠4次免疫后,采用间接ELISA检测血清特异性抗F1-V的IgG和IgA抗体及抗体亚型分类,检测鼻咽喉、肺、小肠及阴道灌洗液中特异性抗F1-V的黏膜分泌型IgA;采用流式细胞术检测鼻相关淋巴组织淋巴细胞、脾淋巴细胞、肠系膜淋巴结及小肠PP结T淋巴细胞表型的变化。结果以rCT-B为佐剂的鼠疫F1-V重组蛋白黏膜疫苗滴鼻免疫后,能够诱导血清中IgG、IgA抗体比正常对照组显著升高（P〈0．01）,同时诱导鼻咽、肺、小肠和阴道内特异性黏膜抗体升高,尤其是肺和生殖道冲冼液内抗体升高极为显著（P〈0．01）。与单纯的F1-V组相比,不同剂量比例疫苗组都能诱导较高、较快的血清IgG、IgA和黏膜sIgA,其中1：2疫苗组能诱导更强的系统免疫和黏膜免疫,但是相比之下,5：1疫苗组是最合适的免疫剂量。结论rCT-B佐剂不仅能提高鼠疫F1-V黏膜疫苗的系统全身免疫应答,还能促进诱导呼吸道、消化道和生殖道等局部黏膜sIgA抗体,增强局部免疫应答,提示rCT-B佐剂能显著提高鼠疫感染的免疫应答作用,这为下一步疫苗的免疫保护评价奠定了基础。     

鼠伤寒沙门氏菌SOD不同途径免疫小鼠的效果观察

目的：观察用鼠伤寒沙门氏菌SOD作为抗原，由不同途径免疫小鼠的抗体产生情况。方法：Balb/c小鼠按免疫途径分为4组--腹腔注射、灌胃、滴鼻与肌注组，各组抗原量相同。于末次免疫后第10、15、20和30天采血，第30天时杀鼠取肠液，用间接ELISA法检测IgA及IgG效价。结果：黏膜外途径免疫小鼠以IgG应答为主；黏膜途径免疫则可建立较好的局部粘膜应答，其中滴鼻组还可引起较好的全身性应答。结论：小鼠可以通过黏膜免疫建立有效的抗鼠伤寒沙门氏菌SOD免疫力。     

表达轮状病毒VP7基因重组腺病毒载体的构建及免疫活性研究

目的 包装表达轮状病毒VP7基因的重组腺病毒,并检测其免疫活性.方法 RT-PCR扩增病毒结构蛋白VP7基因,定向克隆于腺病毒穿梭质粒pAdtrack-CMV中,在细菌中与缺陷型腺病毒基因组pAdeasy-1进行同源重组,并用RT-PCR和Western blot检测.将包装好的腺病毒免疫小鼠,应用间接ELISA检测小鼠血清中特异性轮状病毒IgG抗体.结果 酶切和测序鉴定证实成功构建携带VP7基因的重组腺病毒表达载体,并在293细胞中成功包装病毒;RT-PCR和Western blot 均能特异检测VP7基因的表达;重组腺病毒免疫小鼠后可诱导针对轮状病毒的特异性免疫.结论 重组腺病毒的成功包装,为新型轮状病毒基因疫苗的研制提供了一种可行的途径.     

不同免疫途径对chitosan-DNA疫苗诱导CVB3特异性免疫应答的影响及其意义

目的：确定新型chitosan-DNA疫苗的有效免疫途径。方法：将chitosan-pcDN3-VPI疫分别苗以肌注、口服、滴鼻3种免疫方式免疫Balb/c小鼠；以ELISA检测免疫小鼠血清中IgG、IgM、、IgA，评估其特异性体液免疫应答；以特异性淋巴细胞增殖反应和CTL活性反映其诱导细胞免疫；以5LD50致死剂量CVB3攻击免疫小鼠，评价不同免疫途径的免疫保护效果。结果：①在诱导CVB3特异性体液免疫方面：chitosan-pcDNA3-VPI疫苗肌注组诱生了高水平IgM和IgG，但未能诱生黏膜IgA；口服免疫组仅诱生低水平的黏膜IgA，未能诱生特异性IgM和IgG；滴鼻组可诱生低水平的I埘及高水平的IgG和黏膜IgA。②在诱导CVB3特异性细胞免疫方面：仅滴鼻组诱导了较高水平的淋巴细胞特异性增殖反应和CTL活性；口服组的淋巴细胞增殖活性和CTL活性稍弱；肌注组几乎不能诱导特异性细胞免疫应答。③免疫保护作用：滴鼻组可保护33．3％小鼠长期存活；口服组仅达到16．7％的保护率；肌注组无保护作用。结论：滴鼻免疫途径可能是chitosan-pcDNA3-VPI基因疫苗最合适的诱导全面免疫应答的免疫途径。     

双价志贺疫苗滴鼻免疫小鼠后粘膜免疫与系统免疫应答持续时间的观察

本文探讨双价志贺疫苗滴鼻免疫小鼠一段时间后,粘膜免疫和系统免疫应答的变化。将BALB/c小鼠随机分为三组,每组30只。PBS、FSM-2117和FS-5416(菌量为5×10~6、1×10~7、4×10~7和4×10~7CFU/只/次)经滴鼻途径免疫小鼠。间隔2周,4次免疫后7、30和90d活杀,收集鼻咽、肺、肠、生殖道冲洗液和血清。采用ELISA法检测其中特异性抗福氏、宋内LPSIgA和IgG。结果是两株疫苗经鼻内免疫后,诱发鼻咽、肺、胃肠道和生殖道等不同粘膜部位及血清中特异性抗福氏、宋内LPSIgA、IgG的显著增加(P<0.01)。特异性抗体水平虽然在免疫后30、90d明显下降,但仍明显高于PBS对照组水平。故认为两株双价志贺疫苗滴鼻免疫小鼠后能有效诱导粘膜免疫和系统免疫应答,并持续较长时间。     

重组幽门螺杆菌尿素酶B亚单位疫苗鼻腔免疫的实验研究

目的:探讨基因工程疫苗Hp重组尿素酶B亚单位(rUreB)鼻腔接种的免疫效果。方法:以rUreB不同剂量或加不同佐剂滴鼻免疫BALB/c小鼠。末次免疫7 d后,收集血清及胃黏膜、小肠黏膜、鼻黏膜及气管黏膜冲洗液,用ELISA法检测抗rUreB特异性抗体。结果:rUreB鼻腔免疫后各实验组血清特异性IgG及各黏膜冲洗液中特异性IgA的水平均明显增高,与对照组相比较差异显著(P<0.01)。20μg剂量组与10μg剂量组相比较,仅血清特异性IgG水平增高,其它黏膜特异性IgA的水平未见增高。大肠杆菌不耐热肠毒素B亚单位(LTB)的佐剂效果较霍乱毒素B亚单位(CTB)强,卡泊波可增强鼻腔接种疫苗在胃黏膜洗液中的抗体应答水平。结论:CTB、LTB、卡泊波均可作为rUreB鼻腔黏膜接种的佐剂。HprUreB鼻黏膜接种,不仅可诱导血清特异性抗体反应,而且能引起多个黏膜部位的免疫应答,是一种方便、有效、廉价的免疫途径。     

Efficient intranasal immunization of newborn mice with recombinant adenovirus expressing rotavirus protein VP4 against oral rotavirus infection

Efficacy of passive protection of newborn mice against rotavirus infection by the rotavirus VP4 protein expressed by an adenoviral vector in mice was studied. The VP4 gene was inserted into the E1 region of adenoviral vector pJM17. Recombinant adenovirus Ad5N/VP4 was grown in 293 cells. Intramuscular (i.m.), oral or intranasal (i.n.) immunization of newborn mice with Ad5/VP4 resulted in appearance of VP4-specific antibodies. Specific IgG antibodies were detected in the serum and intestine specimens of i.m. vaccinated mice. Oral immunization elicited serum IgG antibodies and intestinal IgG and IgA antibodies. Compared with i.m. and oral applications, i.n. immunization led to higher levels of serum IgG and intestinal IgG and IgA antibodies. Pups were challenged twice with simian rotavirus SA11 strain orally at the days 7 and 8 after birth. Pups born to i.n. immunized dams achieved 100% protection from rotavirus-induced diarrhea after both challenges. The protection of pups born to orally immunized dams was 80%, while only 30% of pups born to i.m. immunized dams were protected after both challenges. I.n. immunization was most efficient in inducing rotavirus VP4-specific serum, intestinal and milk IgG or IgA in mice that protected newborn mice completely.     

Mucosal administration of completely non-replicative vaccinia virus recombinant Dairen I strain elicits effective mucosal and systemic immunity

We studied the immunogenicity of completely replication-deficient vaccinia virus Dairen I strain recombinant encoding simian immunodeficiency virus (SIV) gag/pol (rDIs) in both mucosal and systemic compartments. When administered either intranasally or intragastrically, rDIs elicited enhanced levels of both SIV Gag p27-specific IgA antibodies and specific plasma antibodies, and the enhanced immunity persisted for the 1-year of observation by intranasal immunization. Increases were observed in antigen-specific IgA antibody-forming cells (AFC) in intestinal mucosal tissues and in IgG AFC in spleens. Furthermore, induction of type 1 and 2 helper cytokines in CD4⁺ spleen T cells and of CD8⁺ IFN-γ spot-forming cells in mucosal tissues was observed in the intranasally immunized mice. Moreover, not even high-dose rDIs generated an SIV gene signal in the brain tissues of immunized mice. These findings suggest that mucosal immunization with the DIs recombinant hold promise as a safe mucosal vector.     

Intranasal Immunization with Heat-Inactivated Streptococcus pneumoniae Protects Mice against Systemic Pneumococcal Infection

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated Streptococcus pneumoniae type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.     

Immunoprophylaxis of Chlamydia trachomatis lymphogranuloma venereum pneumonitis in mice by oral immunization.

Groups of BALB/c mice were orally immunized with chlamydiae and challenged intranasally to determine whether oral immunization offers protection against pulmonary disease and to characterize the nature and kinetics of the chlamydial antibody response in the lung and other mucosal sites. Protection by oral immunization from chlamydial lung disease was demonstrated by lack of replication of the organism and the lack of chlamydial antigen in lung tissue. The chlamydial immunoglobulin A (IgA) antibody response was present at all body sites, reaching peak levels in the lung as well as in the serum. Classical IgA booster effect kinetics was observed after intranasal challenge, especially in the lung. Specific IgG antibody was detected at all body sites but at lower levels. Furthermore, animals immunized orally had no pneumonic process, as determined by histopathology. These studies also suggest that passively acquired specific serum IgG antibody may not significantly influence the course of mucosal replication of the organism. These observations indicate that oral immunization activating the gut-associated lymphoid tissue system gave total protection against chlamydial lung disease, suggesting migration of immunologically competent cells from the intestine to the lung.     

Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates.

This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations. Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB. Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations. Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested. Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung. Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations. Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations. This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization.