钙纳米颗粒作为血吸虫病抗独特型抗体疫苗佐剂的研究

目的 探索钙（Ca）纳米颗粒作为日本血吸虫病抗独特型抗体NP30疫苗佐剂的可行性。方法 将钙纳米颗粒与NP30制备成Ca-NP30结合物（Ca-NP30)，主动免疫BALB/c小鼠，观察其对小鼠的保护性作用并探讨其免疫保护机制。结果 Ca纳米颗粒可增强NP30对宿主的保护性作用，减虫率明显提高，从单用NP30的30.4%提高到57.8%；血清特异性抗体IgG水平较对照组显著升高；足垫试验可引发迟发型变态反应。结论 Ca纳米颗粒可作为血吸虫病抗独特型抗体NP30疫苗的佐剂，其作用机制与同时引起宿主体液免疫和细胞免疫应答增强有关。     

重组IL-2、IL-6、TNF-α作为血吸虫病疫苗佐剂的研究

目的：观察重组IL－2、IL－6和TNF－α（rIL-2,rIL-6,rTNF-α）作为血吸虫病抗独特型抗体疫苗佐剂的可行性，并探讨其作用机制。方法：日本血吸虫抗独特型抗体NP30主动免疫BALB／c小鼠，分别联用rIL-2,rIL-6和rTNF-α，观察其对小鼠的保护性免疫作用，以及对宿主体液免疫和细胞免疫的影响。结果：rIL-2和rIL-6能明显提高NP30疫苗对小鼠宿主的保护性作用，减虫率和减卵率均明显提高，减虫率从单用NP30的40．6％分别提高到53．5％和55．7％，减卵率从46．5％分别提高到68．7％和69．8％；二者均使血清特异性IgG、IgG1和IgG2a抗体明显升高；另外，足垫试验结果显示NP30加用rIL-2可引发迟发型变态反应。rTNF-α无增强NP30的保护性免疫作用。结论：rIL-2和rIL-6可作为血吸虫病抗独特型抗体疫苗的佐剂，其作用机制与同时增强Th1和Th2免疫应答有关。     

日本血吸虫抗独特型抗体NP30抗体检测法在云南大山区应用效果的评价

目的评价日本血吸虫抗独特型抗体NP30抗体检测法在云南大山区血吸虫病流行现场的应用效果.方法对云南大山区血吸虫病流行区的506位居民进行粪检,同时用NP30抗体检测法和血吸虫抗体ELISA检测试剂盒(SEA-ELISA)分别对其血清进行检测,评价NP30抗体检测法的敏感性;同时检测非流行区的100份血清确定其特异性.结果 NP30抗体检测法和SEA-ELISA法的粪检阳性符合率分别为87.80%(144/164)和84.76%(139/164);两者的特异性分别为96.00%(96/100)和94.00%(94/100),差异均无显著性(P均＞0.05),但在粪孵阴性人群血清样本中,NP30抗体检测法和SEA-ELISA法的检出率分别为44.15%(151/342)和70.47%(241/342),差异有显著性(P＜0.05).结论 NP30抗体检测法可用于云南大山区血吸虫病筛查.     

单克隆抗独特型抗体NP30检测78例慢血血清的效果观察

研究证明,无论是在感染血吸虫的小鼠还是人体中,都存在自然产生的抗独特型抗体(anti-id),单克隆抗独特型抗体NP 30即是用生物技术制备抗原-抗独特型抗体NP30作为探针,用双“抗原”夹心酶标法检测血吸虫病人血清中的短程抗体。近来,我们用     

日本血吸虫单克隆抗独特型抗体NP30重链(CDR3)_6基因的构建表达及初步鉴定

目的设计、构建日本血吸虫单克隆抗独特型抗体NP30重链(H链)CDR3区6倍重复表位基因(CDR3)6,并对表达产物进行初步鉴定.方法克隆NP30 H链(CDR3)6基因,导入PET-28(a)载体并在E.coli BL21中表达;表达产物经纯化后用ELISA方法测定活性.结果(CDR3)6基因被成功构建、表达,表达产物经ELISA检测证实其可与抗NP30抗体NP48及日本血吸虫病人血清反应.结论(CDR3)6部分保留了抗独特型抗体表位的亲和性和特异性.     

日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体的构建表达与初步鉴定

目的构建和表达日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体,初步鉴定表达产物的活性。方法用overlap PCR法扩增双链抗体基因VH-GGGGS—VL将双链抗体基因重组入原核表达载体pBAD／gⅢ。表达质粒转化E．coli TOP10F’,左旋阿拉伯糖诱导表达。对表达产物进行分离纯化,ELISA检测纯化蛋白与血吸虫病人血清抗体的结合活性。结果测序证实双链抗体基因正确,构建了双链抗体的原核表达系统,双链抗体在细菌超声上清和沉淀内均有表达,分子量约为34kD。纯化产物经ELISA鉴定,结果表明NP30单特异性双链抗体可与血吸虫病人血清抗体特异性结合。结论构建和表达的日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体具有与亲本单抗相同的结合活性。     

日本血吸虫单克隆抗抗独特型抗体NP41的建株及初步鉴定

目的 制备日本血吸虫单克隆抗独特型抗体（anti-id,Anb2)NP30的抗抗独特型抗体（anti-anti-id,Ab3)。方法 IRS－NP30主动免疫BALB／c小鼠,取其脾细胞与SP2／0细胞融合,制备anti-anti-id杂交瘤细胞株。结果 获得1株稳定分泌单克隆抗体的细胞株,定名为NP41。NP41的Ig同型为IgM。免疫组化显示NP41与成虫的体膜、消化管上皮、子宫内膜上皮及虫卵内皮的毛蚴头腺,毛蚴膜呈阳性反应。结论 NP41为NP30的anti-anti-id,其识别的抗原即NP30的模拟抗原。     

日本血吸虫NP30抗体检测方法的优化

目的为了提高单克隆抗独特型抗体NP30抗体检测系统在大山区日本血吸虫病疫区的诊断效能,在原有的双抗体夹心ELISA方法的基础上对其进行优化、改良,建立间接ELISA血清学诊断方法。方法通过正交设计,选用L27(313)正交表,对NP30腹水包被酶标板的浓度、血清浓度、血清孵育时间和辣根过氧化物酶标记抗人IgG的二抗孵育时间共4个因素的3个水平进行不同组合的实验,确定该间接法的最佳实验条件。并用建立的方法对50份云南大山区日本血吸虫粪孵阳性病人血清样本和50份非疫区正常人血清样本进行检测,评价其敏感性和特异性。结果确定的该间接法最佳检测条件为:NP30腹水4μg/ml包被酶标板,血清浓度为1∶100,37℃孵育60min,二抗为37℃孵育30min。该方法的敏感性和特异性分别为88%和96%。结论该方法具有较高的诊断价值,可用于大山区日本血吸虫病疫区的诊断检测。     

抗人膀胱癌抗独特型抗体佐剂疫苗的动物实验研究

目的 探讨抗独特型抗体佐剂疫苗对膀胱癌的免疫治疗作用。方法 用抗人膀胱癌抗独特型抗体佐剂疫苗(Ab2 SAF)免疫BALB／C小鼠，即实验组；对照组用生理盐水，方法同实验组。免疫3次后，于末次免疫后1w，将新鲜人膀胱移行细胞癌组织移植于小鼠肾包度下，分别于移植后第2、4、6、8和10天处死小鼠，取血清进行抗抗独特型抗体(Ab3)分析。移植瘤行组织学检查，观察宿主淋巴细胞浸润情况及瘤细胞可见率。结果 实验组小鼠肾脏包膜下人膀胱癌细胞系很快受到排斥，在移植后第6天，淋巴细胞浸润即达高峰，而对照组在第10天才达高峰；癌细胞可见率，在移植后第6天即明显降低，而对照组呈逐渐下降。实验组Ab3为阳性，而对照组阴性。结论 Ab2作为抗原免疫小鼠后，通过诱发或激活对人膀胱癌特异性体液和细胞免疫反应，排斥癌细胞并抑制其生长。     

日本血吸虫单克隆anti—anti—id的建株及初步鉴定

日本 制备日本血吸虫抗独特型抗体（anti-id,Ab2)NP30的单克隆抗独型抗体（anti-anti-id,Ab3)。方法 应用辣根过氧化物酶标记的NP30（HRP－NP30）免疫BALB／c小鼠的脾细胞与SP2／0融合,制备单克隆anti-anti-id。结果 获得1株稳定分泌抗NP30的单克隆抗体的细胞株,定名为NP48。NP48的Ig同型为IgG2b。NP48与SWAP和SEA ELISA反应均阳性。免疫组织化学显示NP48可定位于虫卵内毛蚴头腺及毛蚴膜。结论 NP48是NP30的特异性anti-anti-id,可用于NP30模拟抗原的分离和鉴定。     

Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharide of their lipopolysaccharides.

Immunoprophylaxis for bacterial enteric diseases is hindered because the protective immune mechanism(s) against nontyphoidal salmonellae or shigellae in humans are not established. On the basis of the similarities between the clinical signs, epidemiology, pathogenesis, and pathology of as well as protective immunity to salmonellae and shigellae, we propose that serum IgG antibodies to the O-specific polysaccharide (O-SP) of their lipopolysaccharides (LPSs) will confer protective immunity to these two pathogens. Critical to this notion is that (1) the virulence of these two pathogens requires full expression of their LPS; (2) active or passive immunization with serum IgG O-SP antibodies confers protection of mice against Salmonella typhimurium (there are no comparable data for humans); and (3) in humans, convalescence from shigellosis confers type (O-SP) -specific protective immunity, and indirect evidence shows a correlation between the level of serum LPS antibodies and resistance to shigellosis. We designed conjugate vaccines to elicit high levels of long-lived serum IgG O-SP antibodies to nontyphoidal salmonellae and shigellae to test this hypothesis.     

Serum samples from infants vaccinated with a pneumococcal conjugate vaccine, PncT, protect mice against invasive infection caused by Streptococcus pneumoniae serotypes 6A and 6B

Streptococcus pneumoniae serogroup 6 is an important cause of respiratory tract disease worldwide. Vaccination with 6B polysaccharide induces antibody response to the cross-reacting serotype 6A, but the protective capacity of 6A antibodies induced in infants remains unknown. In this study, passive immunization with serum samples obtained from infants vaccinated with an octavalent polysaccharide protein conjugate vaccine, PncT, protected mice against bacteremia and/or lung infection caused by intranasal challenge with serotypes 6B and 6A. Protective infant serum samples had significantly higher serotype-specific IgG levels and opsonic activity than did nonprotective serum samples. The protective level to either serotype was approximately 1 microg of specific IgG antibodies injected per mouse (corresponding to approximately 0.3 microg/mL). The protection was strongly related to opsonophagocytic antibody levels measured in vitro. These results demonstrate that PncT induces antibodies in infants that protect mice against invasive disease caused by the homologous serotype and by the cross-reacting serotype 6A.     

Expression, characterization and therapeutic efficacy of chimeric Fab of anti-idiotypic antibody NP30 against Schistosoma japonicum

The murine monoclonal anti-idiotypic antibody NP30 is a promising therapeutic antibody against Schistosoma japonicum. However, the immunogenicity of murine NP30 limits its further study and application in humans. Here the chimeric Fab of NP30 (chFab-NP30) comprising the variable regions of murine NP30 and constant regions of human antibody was assembled. chFab-NP30 was expressed and purified as a soluble and functional protein. Administration of chFab-NP30 in vivo increased the survival rate, reduced egg burdens and ameliorated organ pathology of mice with acute schistosomiasis. Our study indicated that chFab-NP30 is a promising candidate to be used as a specific and efficient recombinant antibody against acute schistosomiasis japonica. Further studies on function mechanism of chFab-NP30 needs to be carried out in the future.     

日本血吸虫抗独特型抗体NP30对急性血吸虫病的免疫治疗作用

目的探讨日本血吸虫抗独特型抗体NP30对急性血吸虫病的免疫治疗作用。方法根据L9（3^4）正交表设计动物实验,日本血吸虫尾蚴感染C57BL／6小鼠后注射抗独特型抗体NP30,观察不同治疗方案各组小鼠的存活时间并计算不同时问段的生存率,取小鼠肝脏石蜡切片,HE染色,测量小鼠单个虫卵肉芽肿的直径及面积。结果各实验组小鼠的生存中位数为45～78d,其中第4组生存中位数为71d,比同样感染40条尾蚴的对照组2（53d）延长了33．96％。Cox回归分析显示小鼠存活时间主要与尾蚴感染水平、抗体注射途径有关（P均〈0．05）。各实验组小鼠单个虫卵肉芽肿平均直径为179．07～226．86μm,平均面积为（32．11～5t．37）×10^3μm^2;对照组各组小鼠单个虫卵肉芽肿平均直径为205．89～239．86μm,平均面积为（44．61～57．24）×10^3μm^2。极差分析及方差分析均显示抗独特型抗体NP30的注射方式和注射剂量是影响虫卯肉芽肿平均直径和面积的主效应因素。NP30的最佳免疫治疗方案为感染尾蚴28d后,以每鼠每只20μg的剂量连续3次肌肉注射。结论抗独特型抗体NP30可改善血吸虫感染小鼠的生存状况,降低血吸虫对宿主造成的免疫病理损害,具有治疗急性血吸虫病的潜能。     

抗独特型抗体NP30对血吸虫病虫卵肉芽肿及肝纤维化的调节作用

目的　研究单克隆抗独特型抗体NP30主动免疫对血吸虫病虫卵肉芽肿及肝纤维化的影响。方法　实验组ICR小鼠腹腔注射NP30 10 0 μg/次 ,连续免疫 3次 ,对照组腹腔注射SP2 / 0腹水。尾蚴攻击感染后第 4、8、12、16、2 0和 2 4周分别处死小鼠剖取肝脏 ,用VG(VanGieson)组织化学染色 ,Ⅰ型胶原、Ⅲ型胶原和纤维连接蛋白 (FN)免疫组织化学染色 ,应用计算机图像分析系统对肝脏虫卵肉芽肿体积和虫卵肉芽肿内的胶原沉积进行定量测定。结果　尾蚴攻击感染第 12周后 ,实验组虫卵肉芽肿的体积明显小于对照组 ,虫卵肉芽肿细胞组分与对照组明显不同 ,出现两种不典型肉芽肿。VG染色显示实验组虫卵肉芽肿内胶原的平均光密度值明显小于对照组。免疫组织化学显示实验组虫卵肉芽肿内Ⅰ型胶原、Ⅲ型胶原及FN含量均比对照组低。结论　NP30接种可能诱导体液和细胞两种保护性免疫 ,对血吸虫病虫卵肉芽肿具有负调节作用 ,对血吸虫性肝纤维化有明显的抑制作用     

Intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity.

The use of interleukin (IL)-12 as a new vaccine adjuvant for stimulating protective antiviral mucosal immunity has been examined. Mice were immunized intranasally (in) with an influenza vaccine consisting of soluble hemagglutinin (H1) and neuraminidase (N1) plus IL-12. This treatment resulted in elevated levels of lung and splenic interferon-gamma and IL-10 mRNA. Total and IgG2a anti-H1N1 antibody levels in serum were significantly elevated, as were total, IgG1, IgG2a, and secretory IgA antibody levels in bronchoalveolar lavage (BAL) fluids compared with animals receiving vaccine alone. Mice immunized in with vaccine and IL-12 also exhibited decreased weight loss and dramatically enhanced survival after lethal challenge with infectious influenza virus. Protection was dependent upon the presence of B cells and could be transferred to naive mice by inoculation of either serum or BAL fluid from IL-12-treated mice. These findings show for the first time that soluble IL-12 delivered in serves as a powerful respiratory adjuvant for protective antiviral immunity.     

Characterization of the serum antibody response to the capsular polysaccharide of Haemophilus influenzae type b in children with invasive infections.

The serum antibody response to the capsular polysaccharide of Haemophilus influenzae type b (Hib) was studied in 30 children aged 1 day-5 years with invasive Hib infections. From each child, serum was obtained 0-2 days, 5-11 days, 1 month, and 6-12 months after onset of symptoms. Total antibodies were determined with RIA and isotypes with ELISA. Only 2 children had antibody levels above the estimated protective level (0.15 microgram/mL) in the first serum sample. The antibody response was age dependent with wide individual variations. Children > or = 2 years had increases in IgG, IgM, and IgA antibodies with predominance of IgG. The initial IgG response was IgG1 and IgG2 with predominance of IgG1. In the last serum sample, IgG1 antibodies had decreased while IgG2 antibodies remained unchanged. Only 2 of 7 children < 1 year had a detectable antibody response. The correlation coefficient for total antibodies compared with the sum of IgG, IgM, and IgA was .88 (P < .0001) and for IgG compared with the sum of IgG1 and IgG2 was .97 (P < .0001).     

Induction of immunity in mice to Fasciola hepatica with a Fasciola/Schistosoma cross-reactive defined immunity antigen

The paradox of schistosomiasis is that infection confers immunity to its host, yet immunization with subcellular antigens of the parasite does not, in general, induce protective immunity. Infection or immunization with subcellular antigens of Fasciola hepatica confers high levels of immunity to a challenge infection with another trematode, Schistosoma mansoni. We have isolated by antibody affinity chromatography a Fasciola hepatica/Schistoma mansoni cross-reactive antigen, designated FhSmIII(M), and also have shown that this antigen confers immunity in mice to a challenge infection with S. mansoni. This antigen was compared with a crude F. hepatica worm extract (FhWWE) as to its ability to induce an IgG antibody response in mice, and to determine whether it had a protective effect in mice to a challenge infection with F. hepatica metacercariae. Mice immunized with FhSmIII(M) or FhWWE, and subsequently infected with F. hepatica, developed higher IgG antibody levels to FhSmIII(M), as measured by ELISA, than F. hepatica-infected controls. Mice immunized with FhWWE did not develop significant levels of resistance to challenge with F. hepatica metacercariae. Mice immunized with FhSmIII(M) and infected with F. hepatica metacercariae developed 69%-78% less worms than controls. An F. hepatica/S. mansoni cross-reactive, cross-protective defined immunity antigen confers in mice significant levels of protection to a challenge infection with F. hepatica.     

Induction of opsonic antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide by an anti-idiotypic monoclonal antibody.

Mucoid strains of Pseudomonas aeruginosa are the major pulmonary pathogens for cystic fibrosis patients. Opsonizing antibodies to the mucoid exopolysaccharide (MEP) antigen may protect animals and some cystic fibrosis patients from infection. However, MEP does not readily elicit opsonic antibodies either during chronic infection or after vaccination. To evaluate alternative means to induce opsonic antibodies, a murine monoclonal anti-idiotypic antibody directed to an opsonic monoclonal antibody specific to MEP was produced. The anti-idiotypic antibody bound to F(ab')2 fragments of the opsonic antibody, blocked binding to MEP, bound to cross-reactive idiotopes on human opsonic antibodies to MEP, and elicited MEP-specific antibodies in syngeneic and allogeneic mice. These anti-idiotype-induced, MEP-specific antibodies fixed complement to mucoid P. aeruginosa cells and opsonized them for phagocytic killing by human leukocytes. These studies demonstrate the potential utility of anti-idiotypic monoclonal antibody for generating protective immunity against bacterial polysaccharides.