O1或O139霍乱疫苗对异群霍乱缺乏交叉保护

O1和O139霍乱弧菌是引起急性腹泻的病原微生物,用这两群菌的灭活全菌体与重组霍乱毒素B亚单位构建的亚单位／菌体型疫苗免疫队CA小鼠,对本群菌的攻击可提供良好免疫保护,而对异群霍乱菌则缺乏交叉保护作用。     

肠道疫苗保护作用的免疫相关物

正保护作用的免疫相关物(CoP)是一种免疫应答,这种应答在统计学上与保护作用相关联。免疫相关物可能是机制性的,也可能是非机制性的,对一种疾病而言可能有一种以上的免疫相关物,它们通常被称为共相关物。大多数目前已知的免疫相关物与中和血清或黏膜抗体有关,在个别情况下抗体的其他功能可能更加重要。此外,细胞免疫应答常常与抗体起协同保护作用。目前已经批准上市了一些抗某些肠道病原体的疫苗,还有一些抗其他病原体的候选疫苗正处在研     

rBS-WC(O139)霍乱疫苗的小鼠免疫试验

1992年起在印度和孟加拉国相继发生的O139霍乱,目前已迅速传播到其周边国家,对我国亦已构成严重威胁。当前全世界预防霍乱的工作更加复杂,除了要针对O1群霍乱弧菌研制保护期长、保护效率高的疫苗外,还要对这一新发现的时隐时现的烈性腹泻病原菌快速作出反应,及时分析其保护性抗原,构建有效疫苗,以控制疫情发展。 O139霍乱弧菌与O1群霍乱弧菌有很大不同,特别在菌体抗原方面存在显著差异。O1群菌感染的患者康复后对同群菌的冉感染有免疫保护作用,而对O139群菌的感染则无此保护作     

O1群霍乱Ogawa血清型结合疫苗免疫学效果研究

霍乱是经粪口传播的烈性传染病,相应的疫苗研究已逾百年,但目前还没有理想的疫苗。实验中以霍乱O1群小川血清型的脂多糖为目标抗原,用不同方法制备了其四种霍乱结合疫苗,通过小鼠模型验证了各结合物的免疫学效果。结果显示,不同结合物免疫学效果不一,其中增大多糖分子量后制备的结合物免疫效果较好,氨还原法制备的结合物多针免疫后也可诱导特异性抗体产生,而且具有针对小川和稻叶两种血清型的杀弧菌活性。     

金黄色葡萄球菌a-溶血素亚单位疫苗在小白鼠模型的免疫效力评价

目的:以小白鼠为试验模型,检测金黄色葡萄球菌α 溶血素基因工程蛋白单位疫苗的抗体效价水平和免疫保护力,初步评价此亚单位疫苗的免疫效力。方法:Bradfrod法对目的蛋白进行蛋白含量测定,Geoffroy法测定目的蛋白半数溶血效价和活性,建立ELISA方法测定抗体效价,并且通过攻毒得出疫苗免疫保护力。结果:纯化的目的蛋白在53kDa处显示单一条带,蛋白含量为0.1278mg/ml;溶血比活为8012.5HU/mg;所有试验小鼠在首免一周后采集的血清中都检测到了特异性抗体,而抗体效价开始时呈逐渐上升趋势,达最高值后随时间延长逐渐降低。结论:纯化的目的蛋白达到电泳级纯度,且依然保持良好的黏附活性,蛋白疫苗作用机体产生的抗体水平符合抗体消长规律。     

霍乱O139血清群多糖结合疫苗的生物合成研究

目的:霍乱是由霍乱弧菌引起的一种烈性传染病,其防治已成为一个全球性的公共卫生问题。其中1992年出现的O139血清群是除O1外另一种病原体,发病数日益增多。因此,有必要寻找一种安全有效适用于各种人群的疫苗。方法:敲除霍乱弧菌O139血清群93-3株脂多糖合成途径中O抗原连接酶基因waa L,在周间质产生游离的多糖,之后转入包含来自脑膜炎奈瑟球菌的糖基转移酶和霍乱毒素B亚单位(CTB)编码序列的共表达载体,经IPTG诱导后制备全菌蛋白样品,利用抗His抗体检测糖蛋白的表达,利用Ni柱和离子交换柱对糖蛋白进行纯化,并对其进行糖定量和蛋白定量。结果:以未糖基化、相对分子质量约为14×103的底物蛋白CTB为对照,当共表达CTB和糖基转移酶Pgl L时,通过Western印迹可检测到相对分子质量约为20×103的糖基化蛋白,经Ni柱及阳离子交换柱纯化,得到纯度较高的O139群霍乱O抗原多糖结合蛋白,其纯度约为84.2%,并计算得其糖-蛋白比为0.103∶1。结论:通过生物法合成了一种霍乱O139血清群的多糖结合疫苗,为后续进行动物评价打下了基础。     

卵形鲳鲹对刺激隐虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳NFDA8(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力.实验结果显示:两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强.同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致.因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的.鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

禽流感特异性转移因子的制备及其免疫作用

目的制备禽流感病毒特异性转移因子并探讨其对禽流感灭活疫苗的免疫增效作用。方法用禽流感病毒H5N1血清亚型灭活疫苗免疫鸡,用国标血凝抑制方法检测病毒特异性血凝抑制抗体效价。当抗体效价达到高峰时,翅静脉采取外周血,分离淋巴细胞并制备细胞单层、传代后获得禽流感病毒H5N1血清亚型特异性转移因子。用所获得的特异性转移因子进行疫苗免疫增效试验。结果采用本法可获得禽流感病毒特异性转移因子。免疫增效试验表明,在进行禽流感病毒灭活疫苗免疫的同时使用禽流感病毒特异性转移因子,可在一定幅度内提高禽流感病毒抗体水平并能延长抗体维持时间。不同给药途径比较试验表明,口服途径给药的疫苗增效作用优于注射途径给药。结论通过淋巴细胞体外培养可以制备禽流感病毒特异性转移因子。禽流感病毒H5N1血清亚型特异性转移因子对禽流感病毒灭活疫苗具有明显的增效作用,且口服途径给药的疫苗免疫增效作用优于注射途径给药。     

吸附霍乱菌苗免疫后人群血清杀弧菌抗体观察

本文介绍了用霍乱杀弧菌抗体微量法,检测经Ｏ１型吸附霍乱菌苗免疫前后人群的Ｏ１型霍乱和Ｏ１３９型霍乱的血清杀弧菌抗体。结果表明,免前人群的Ｉｎａｂａ、Ｏｇａｗａ、Ｏ１３９型三种自然杀弧菌抗体很低（ＧＭＴ在５．４９以下）,免后Ｉｎａｂａ和Ｏｇａｗａ型杀弧菌抗体大幅度增加,ＧＭＴ分别达到７８２和５９２、Ｏ１３９型霍乱杀弧菌抗体免疫前后无变化     

维氏气单胞菌菌蜕疫苗免疫后鲤免疫应答与hepcidin基因表达特征

【目的】为探讨接种维氏气单胞菌菌蜕疫苗后鲤的应答反应特征及与甲醛灭活苗的效果比较,在实验条件下研究了接种疫苗后鲤(Cyprinus carpio)的特异性、非特异性免疫指标及hepcidin基因表达量的变化。【方法】在开始实验的第1、15和29天使用菌蜕疫苗(CLGs)和甲醛灭活疫苗(FKC)对鲤进行注射3次免疫,并设一个注射磷酸缓冲液(PBS)的对照组,每个组设3个重复。从第一次免疫后每隔7 d从每个重复中随机取三尾鱼进行取样,对血清抗体滴度、相对免疫保护率(RPS)、溶菌酶(LZM)、呼吸暴发、补体C3、髓过氧化物酶(MPO)、丙二醛(MDA)及hepcidin基因表达量进行测定。【结果】二次免疫后,CLGs组抗体凝集效价达2~6-2~8,显著高于FKC和PBS组(P0.05),且其呼吸暴发、LZM、MPO含量和hepcidin基因表达量显著高于PBS组(P0.05),MDA含量显著低于PBS组(P0.05);CLGs组C3含量在35和42 d显著低于PBS组(P0.05);FKC组MPO含量和hepcidin基因表达量显著低于菌蜕组(P0.05)。CLGs组的RPS为57.70%,FKC组RPS为30.77%。【结论】接种一定强度维氏气单胞菌菌蜕疫苗,能够提高鲤血清抗体效价和呼吸暴发、LZM、MPO等非特异性免疫指标及hepcidin基因表达,而且整体免疫效果好于甲醛灭活苗。     

Immune responses of mice to influenza subunit vaccine in combination with CIA07 as an adjuvant

CIA07 is an immunostimulatory agent composed of E. coli DNA fragments and modified LPS lacking the lipid A moiety. In this study, we investigated whether CIA07 promotes immune responses as an adjuvant to the influenza subunit vaccine. Balb/c mice were immunized intramuscularly once or twice at a 4-week interval with the trivalent influenza subunit vaccine antigen alone or in combination with CIA07 as adjuvant. Antigen-specific serum antibody titers and hemagglutination-inhibition (HI) antibody titers were assessed. At 4 weeks after each immunization, the antigen-specific total serum IgG antibody titer in mice receiving CIA07 was 2 to 3 times higher than that in animals administered antigen alone (P<0.05). The CIA07-treated group additionally displayed higher HI antibody titers against each of the 3 vaccine strains, compared to the antigen group. Animals receiving antigen alone displayed barely detectable antigen-specific serum IgG2a antibody titers. In contrast, coadministration of CIA07 with antigen led to significantly enhanced IgG2a antibody responses, suggesting that CIA07 stimulates a Th1-type immune response. Moreover, the CIA07-treated group displayed a marked increase in the number of interferon gamma-producing CD8(+) T cells in splenocytes. These data collectively demonstrate that CIA07 has the ability to induce both Th1-type cellular and Th2-type humoral immune responses to the influenza subunit vaccine, and support its potential as an effective adjuvant to the influenza vaccine.     

汉滩病毒84Fli株DNA疫苗诱导小鼠免疫应答的初步研究

为了加强我国病毒性出血热的防治,本研究将汉滩病毒84Fli株核蛋白S和糖蛋白M编码片段分别克隆至pcDNA3.0载体,构建了pcDNA3/84S和pcDNA3/84M重组质粒,等量混合采用肌肉注射途径免疫C57BL/6小鼠,免疫3次,每次间隔2周,同时与双价出血热病毒灭活疫苗进行对比。ELISA及免疫荧光(IFA)分别检测小鼠血清中汉滩病毒核蛋白及糖蛋白特异性抗体,流式细胞仪和ELISPOT方法分析小鼠免疫后的细胞免疫水平。微量中和试验检测小鼠血清抗体的的中和活性。结果显示,DNA疫苗免疫组C57BL/6小鼠在初次免疫2周后即能检测到汉滩病毒核蛋白与糖蛋白的特异性抗体,与灭活疫苗组相比,重组质粒诱导的抗体滴度高,产生时间早,产生的抗体具有中和活性;同时可诱导产生特异性细胞免疫应答。研究表明,汉滩病毒pcDNA3/84S和pcDNA3/84M重组质粒能有效刺激小鼠产生特异性体液免疫和细胞免疫应答。     

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.     

O2血清型肺炎克雷伯氏菌多糖结合疫苗的生物合成

本研究旨在利用微生物体内合成肺炎克雷伯氏菌 (Klebsiella pneumoniae,Kp) 多糖结合疫苗并研究其保护效果。通过敲除Kp O抗原连接酶基因waaL阻断其LPS合成,再向缺失株中导入糖基工程载体,使细菌能够在体内合成糖蛋白,并将该糖蛋白免疫小鼠后评价其保护效果。结果表明,在构建的 Kp waaL缺失株中导入糖基工程载体后,底物蛋白重组霍乱毒素B亚单位rCTB (Recombinant cholera toxin B subunit) 能够被O糖化,从而得到糖蛋白;动物实验结果显示该疫苗能刺激小鼠产生较高的抗体效价,试验组小鼠攻毒后一周存活率可达75%。这种生物合成方法制备的多糖结合疫苗有望成为针对肺炎克雷伯氏菌的新型候选疫苗。     

Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

Background

Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid marker for assessing vaccination status in a fish population. For this purpose a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate sera from farmed rainbow trout vaccinated against V. anguillarum O1.

Study Design

Immune sera from rainbow trout immunised with an experimental vaccine based on inactivated V. anguillarum O1 bacterin in Freund’s incomplete adjuvant were used for ELISA optimisation. Subsequently, sera from farmed rainbow trout vaccinated with a commercial vaccine against V. anguillarum were analysed with the ELISA. The measured serum antibody levels were compared with the vaccine status of the fish (vaccinated/unvaccinated) as evaluated through visual examination.

Results

Repeated immunisation with the experimental vaccine lead to increasing levels of specific serum antibodies in the vaccinated rainbow trout. The farmed rainbow trout responded with high antibody levels to a single injection with the commercial vaccine. However, the diversity in responses was more pronounced in the farmed fish. Primary visual examinations for vaccine status in rainbow trout from the commercial farm revealed a large pool of unvaccinated specimens (vaccination failure rate = 20%) among the otherwise vaccinated fish. Through serum analyses using the ELISA in a blinded set-up it was possible to separate samples collected from the farmed rainbow trout into vaccinated and unvaccinated fish.

Conclusions

Much attention has been devoted to development of new and more effective vaccines. Here we present a case from a Danish rainbow trout farm indicating that attention should also be directed to the vaccination procedure in order to secure high vaccination frequencies necessary for optimal protection with a reported effective vaccine.     

鼠疫溶菌疫苗免疫小鼠的体液免疫应答

为选择以F1抗原为主要有效成分的鼠疫溶菌疫苗(Whole cell lysate of Yersinia pestis vaccine,WCLY)的免疫程序,设计了这组试验。在37℃培养鼠疫EV菌,通过超声波裂解法制备鼠疫溶菌疫苗。设计(0,2周)、(0,4周)、(0,2,4周)三种免疫程序,以每剂总蛋白量7.9μg、31.5μg和126.0μg三个剂量皮下接种NIH小鼠。分别在第一针免疫后2、4、8、12周采集血清,通过间接ELISA检测抗鼠疫菌F1抗原和总抗原抗体。结果显示:免疫后血清抗体上升很快,2周内即可测出;无论哪种免疫程序,至12周时抗体滴度仍保持高水平;加强免疫后,抗体水平在4周或8周达到较高,可与活疫苗免疫者相比;溶菌疫苗的接种剂量为7.9μg时,动物只出现轻度不良反应。提示鼠疫溶菌疫苗需要两剂免疫,最短可间隔2周,接种剂量应不超过7.9μg,疫苗中应富含F1抗原。     

Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds ( n  = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.     

O139霍乱灭活全菌体菌苗免疫后人群抗体水平测定

Ｏ１３９霍乱弧菌经甲醛灭活制备而成的全菌体菌苗在广西田阳县进行临床考核。每组５０名中学生经肌肉途径接种４５亿菌和９０亿菌两种剂量,一个月后８０％接种者血清中杀弧菌抗体升高,最高可达１∶１６０,免疫后３个月开始缓慢下降,但仍有６８－７２％接种者抗体效价在１∶２０以上。高剂量组半数接种者的杀弧菌抗体可维持存在６个月。血清中凝集抗体升高明显,但３个月后下降也明显。与对照组Ｏ１菌苗相比,两种抗体水平升高与下降趋势一致。采用ＥＬＩＳＡ法测定抗毒抗体,Ｏ１３９菌苗组与Ｏ１菌苗组结果相似,均有一定上升。上述结果提示Ｏ１３９霍乱灭活菌苗可作为短效应急菌苗使用     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.