浓缩地鼠肾细胞狂犬病疫苗与纯化Vero细胞狂犬病疫苗接种 …

为了解国产浓缩地鼠肾细胞狂犬疫苗与法国纯化Ｖｅｒｏ细胞狂犬疫苗接种人体后中和抗体产生情况。分别用两种疫苗接种１０人,用小鼠中和试验方法检测中和抗体滴度。国产疫苗五针全程免疫后３０天（第一针按种后６０天）可１００％达到保护水平,法国疫苗在第一针接种后３０天１００％达到保护水平。第一针接种后１４、３０、６０天时,前者抗体平均滴度分别是０．０６ＩＵ／ｍｌ、１０２ＩＵ／ｍｌ、２．０７ＩＵ／ｍｌ;后者抗体平     

银鲫口服嗜水气单胞菌疫苗的免疫和免疫组化研究

室内恒温条件下(水温24±1℃)分别用微胶囊疫苗与全细胞疫苗口灌免疫银鲫,口灌免疫后每周取血和前、中、后肠肠黏膜,间接ELISA检测其抗体效价。结果发现:微胶囊疫苗组鱼血清抗体效价较高,峰值为1∶80,且维持时间较长;各肠段的黏膜抗体中,后肠黏膜抗体效价最高,微胶囊疫苗组峰值达1∶320,前肠的肠黏膜抗体效价也有一定的水平为1∶40;肠道免疫组化试验也得到相似结果:在不同时间对前、中、后肠切片进行免疫组化染色,从光镜下观察到肠黏膜外层和黏膜下层都有阳性着色,以包裹全菌苗为最多,未包裹菌苗较少。同时进行两组疫苗的池塘网箱免疫试验,结果表明:微胶囊疫苗组与全细胞疫苗组的相对百分成活率分别为61.1%和50%,微胶囊疫苗组略高于全细胞疫苗组;口服免疫后两组鱼血清中均能产生较高的抗体效价,在30d左右抗体效价最高达1∶512,能维持较长时间约8—10个月,但两者差异不显著。     

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。     

鸡马立克氏病毒和网状内皮增生病毒感染肉鸡时的相互作用

为了研究鸡马立克氏病病毒(MDV)和网状内皮增生病病毒(REV)共感染时的相互作用,分别在REV母源抗体阳性(REV-Ab+)和阴性(REV-Ab-)及经MDV疫苗CVI988株免疫和不免疫的商品代肉鸡,比较了二种病毒在病毒血症水平和特异抗体效价上的相互影响.结果表明,在未经CVI988株免疫鸡,REV病毒血症对MDV强毒接种后的病毒血症水平及抗体效价无明显影响,但REV病毒血症显著抑制了CVI988疫苗免疫为鸡提供的抵抗力和抗体效价,因而提高了强毒MDV感染后的病毒血症的程度.另一方面,MDV感染会显著减弱REV-Ab+鸡对REV感染的抵抗力,提高REV-Ab+鸡在感染REV后的病毒血症水平并抑制对它的抗体效价.分析表明,MDV和REV共感染主要通过抑制鸡体的免疫功能来影响另一种病毒的复制及其致病作用.     

鸡马立克氏病毒和网状内皮增生病毒感染肉鸡时的相互作用

为了研究鸡马立克氏病病毒(MDV)和网状内皮增生病病毒(REV)共感染时的相互作用,分别在REV母源抗体阳性(REV-Ab )和阴性(REV-Abˉ)及经MDV疫苗CVI988株免疫和不免疫的商品代肉鸡,比较了二种病毒在病毒血症水平和特异抗体效价上的相互影响。结果表明,在未经CVI988株免疫鸡,REV病毒血症对MDV强毒接种后的病毒血症水平及抗体效价无明显影响,但REV病毒血症显著抑制了CVI988疫苗免疫为鸡提供的抵抗力和抗体效价,因而提高了强毒MDV感染后的病毒血症的程度。另一方面,MDV感染会显著减弱REV-Ab 鸡对REV感染的抵抗力,提高REV-Ab 鸡在感染REV后的病毒血症水平并抑制对它的抗体效价。分析表明,MDV和REV共感染主要通过抑制鸡体的免疫功能来影响另一种病毒的复制及其致病作用。     

乙肝病毒RNA复制子疫苗与DNA疫苗对小鼠免疫效率的比较

为建立并获取更有效的乙肝疫苗,本实验通过将所构建的HBV RNA复制子疫苗和DNA疫苗分别免疫小鼠,检测细胞免疫与体液免疫的效果.结果表明,以pSFV为基础构建的疫苗载体免疫小鼠后采集的血清中抗体效价不随免疫剂量的增加而提高,在较低剂量免疫的时候,RNA复制子疫苗所产生的抗体效价优于DNA疫苗.并且RNA复制子疫苗在以较低剂量免疫后脾细胞CTL活性高于DNA疫苗.本研究证明HBV RNA疫苗比DNA疫苗表达效果更好,安全性更高,更具有应用前景.     

一种狂犬病毒疫苗佐剂的制备及其免疫效果研究

目的:以角鲨烯、山梨醇、吐温为组分,在琥珀酸缓冲液中混和甲型副伤寒沙门菌鞭毛蛋白制备复合佐剂Nano-fla,评价该佐剂对人二倍体狂犬疫苗的免疫效果和安全性。方法:以人二倍体细胞制备的狂犬灭活疫苗为抗原,BALB/c小鼠设置PBS对照组、全剂量疫苗组、半剂量疫苗组、低剂量佐剂组(含半剂量抗原+5μg鞭毛蛋白)、中等剂量佐剂组(含半剂量抗原+10μg鞭毛蛋白),免疫程序为在0、3、7 d肌肉注射,并在首针免疫后的第7、14 d尾静脉采血分离血清,通过快速免疫荧光灶抑制实验检测中和抗体,通过酶联免疫斑点实验检测细胞因子水平。结果:首针免疫后第7 d,中等剂量佐剂组的中和抗体达保护效力水平,显著高于全剂量疫苗组和低剂量佐剂组;第14 d时中等剂量佐剂组的IgG抗体浓度显著高于全剂量疫苗对照组;第14 d中等剂量佐剂组与全剂量疫苗组相比,分泌IFN-γ和IL-4的淋巴细胞数量显著增加。结论:复合佐剂Nano-fla应用到狂犬疫苗中,能有效刺激小鼠体液免疫和细胞免疫应答,更早地产生中和抗体,且能有效降低抗原用量,具有潜在应用价值。     

不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平分析

目的研究伤寒Vi多糖蛋白结合疫苗免疫效果及不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平,以确定合适的免疫剂量。方法将150只清洁级NIH雌性小鼠随机分为5组,分别为A组(0.625μg结合疫苗组)、B组(1.250μg结合疫苗组)、C组(2.500μg结合疫苗组)、D组(2.500μg多糖组)及阴性组(10mmol/L PBS),每组30只;另领取10只为空白对照(不接种)。A、B、C、D组及阴性组小鼠经腹股沟皮下注射,剂量0.1 m L/只,每隔2周免疫1次,共免疫3次,每次免疫后第7天采血。采用ELISA检测小鼠血清抗体效价,同时对不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体效价进行分析比较。结果与D组相比,A、B、C三组诱导的抗体水平与之均有统计学意义(P<0.05);A组与B组、A组与C组之间的抗体水平也具有统计学意义(P<0.05),而B组与C组之间的抗体水平无统计学意义(P>0.05)。说明与多糖疫苗相比,伤寒Vi多糖蛋白结合疫苗能够诱导更高的抗体水平,且具有明显的剂次加强效应。同时证明1.250μg的伤寒Vi多糖蛋白结合疫苗可诱导与2.500μg伤寒Vi多糖蛋白结合疫苗相同的抗体水平。结论伤寒Vi多糖蛋白结合疫苗的两种免疫剂量在小鼠体内可诱导相同的抗体水平,在选择接种剂量时,可致免疫应答的无统计学意义的低剂量可能是较为经济和安全的选择。     

狂犬病毒糖蛋白的克隆表达及其在中和抗体检测中的应用

目的:制备基因工程表达的狂犬病毒糖蛋白优势表位抗原,并评价其在疫苗免疫后中和抗体检测中的应用价值。方法:TRIzol法从狂犬病疫苗中提取总RNA,经RT-PCR获得糖蛋白目的基因片段,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白,以重组蛋白作为包被抗原,初步建立检测糖蛋白中和抗体的ELISA方法。结果:获得狂犬病毒糖蛋白优势表位区段抗原,建立了糖蛋白中和抗体ELISA检测方法。该检测方法对59例健康献血员血浆样本检测特异性为98.31%(58/59),接种狂犬疫苗免疫个体血浆样本抗体阳性率为98.95%(94/95)。结论:基因工程表达的狂犬病毒糖蛋白优势表位抗原可用于人接种狂犬疫苗后疫苗免疫效果评价。     

补体C3对大肠杆菌疫苗的免疫增强作用

从SPF鸡血清中分离纯化C3,用戊二醛将其与大肠杆菌抗原连接,免疫注射SPF鸡;对照组鸡免疫注射弗氏完全佐剂大肠杆菌疫苗。分别在免疫后的第2、3、4、5、6、7、8、9周采血,用ELISA方法测定血清中的抗体含量,同时测定血清中总补体活性。结果表明,免疫后3周,弗氏佐剂大肠杆菌疫苗诱导鸡体产生的抗体效价高于C3佐剂疫苗组,但至第4周,弗氏佐剂疫苗组的抗体水平达到高峰(OD值=0.270±0.004),然后迅速下降,到第9周降至0.200±0.005,而C3疫苗组鸡免疫后的抗体水平持续上升,从免疫后第2周的0.098±0.003上升到第8周的0.275±0.002。证明C3能够促进免疫记忆细胞的产生,并能够使细菌抗原给予免疫细胞持续稳定的刺激,从而使鸡体维持高水平抗体的时间延长。研究结果为研制有效的家禽细菌性疫苗提供了理论依据。     

Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata)

A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U./ml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine.     

Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island,Indonesia

Effective parenteral vaccines are available to control rabies in dogs. While such vaccines are successfully used worldwide, the period between vaccine boosters required to guarantee protection of the population against rabies varies between vaccines and populations. In Flores Island, Indonesia, internationally and locally produced rabies vaccines are used during annual vaccination campaigns of predominantly free-roaming owned domestic dogs. The study objective was to identify the duration of the presence and factors associated with the loss of adequate level of binding antibodies (≥0.5 EU/ml) following rabies vaccination in a domestic dog population on Flores Island. A total of 171 dogs that developed an antibody titre higher or equal to 0.5 EU/ml 30 days after vaccination (D30), were repeatedly sampled at day 90, 180, 270, and 360 after vaccination. On the day of vaccination (D0), an interview was performed with dog owners to collect information on dog characteristics (age, sex, body condition score (BCS)), history of rabies vaccination, kind of daily food, frequency of feeding, and origin of the dog. Serum samples were collected and the level of antibodies was quantitatively assessed using ELISA tests. Dogs were categorized as having an adequate level of binding antibodies (≥0.5 EU/ml) or inadequate level of binding antibodies (<0.5 EU/ml) at each time points examined. A total of 115, 72, 23, and 31 dogs were sampled at D90, D180, D270, and D360, respectively, with the highest proportion of antibodies ≥ 0.5 EU/ml (58%, 95% CI, 49–67%) at D90, which reduced gradually until D360 (35%, 95% CI, 19–52%). Multivariable logistic regression models showed that loss of adequate level of binding antibodies is significantly associated with dogs having no history of vaccination or vaccination applied more than 12 months before D0, being less than 12 months of age, and having a poor BCS. These results highlight the importance of BCS regarding the immune response duration and provide insights into frequency of vaccination campaigns required for the internationally available vaccine used on Flores Island. For dogs without vaccination history or vaccination being applied more than 12 months before D0, a booster is recommended within 3 months (a largest drop of antibodies was detected within the first 90 days) after the first vaccination to guarantee measurable protection of the population that lasts at least for one year.     

Human rabies immunoglobulin assayed by the rapid fluorescent focus inhibition test suppresses active rabies immunization

The rabies antibody content of each of ten lots of human rabies immunoglobulin was titrated by both the mouse neutralization test and the rapid fluorescent focus inhibition test. The two tests did not give comparable results, the antibody titres obtained by the mouse neutralization test being 1·4–9·6 times higher than those obtained by the rapid fluorescent focus inhibition test. This titre difference was associated with a consistently lower antibody response in human volunteers who had received post-exposure rabies vaccine treatment which included the administration of RIG assayed by the RFFIT.     

Immunization of Cattle Against Rabies Using Inactivated Cell Culture Vaccines

Twenty-one heads of cattle were vaccinated with Madibovin, 31 with Rabdomun and 127 with Rabisin on 4 different farms. Rabies neutralizing antibody titre (≥0.5 IU/ml) was detected in 80% of 163 animals tested about 1 month and in 42% of 133 animals tested about 1 year after primary vaccination. On 3 of the farms 86 animals received booster vaccination about 1 year after primary vaccination. All these animals had antibody titre (≥0.5 IU/ml) about 1 month after booster and antibody levels were higher than after the primary vaccination. Rabies antibody titres (≥0.5 IU/ml) were detected in 96% of 50 animals tested 1 year after the booster. No significant differences (p>0.05) in antibody levels were detected between animals vaccinated with Madibovin or Rabisin (farm C) respectively with Rabisin or Rabdomun (farm D) at any collection time. Responses to rabies vaccines varied considerably between the farms. After primary vaccination of the animals on 2 farms with the same batch of Rabisin, the antibody levels clearly differed (p<0.0001) between the farms. Our results indicate that booster is always necessary after primary vaccination to ensure that all animals are protected.     

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.     

A Comparison of Two Serological Methods for Detecting the Immune Response after Rabies Vaccination in Dogs and Cats being Exported to Rabies-free Areas

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre of > or = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre < 1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (> or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.     

Essential Role of T Cells in the Postexposure Prophylaxis of Rabies in Mice

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta-propiolactone-inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.     

Vaccination of School Children With Live Mumps Virus Vaccine

Live, attenuated mumps virus vaccine (Mumpsvax) was administered to 146 school children 6 to 9 years of age. One child developed clinical mumps nine days after vaccination; epidemiological and serological data strongly suggest that this child had become infected before vaccination. Apart from this single instance there were no apparent clinical reactions that could be ascribed to the administration of the vaccine. Sixty-three of the 146 children with no clinical history of mumps had an initial serum neutralizing antibody titre of less than 1:2. Specific antibodies to mumps virus were detected in 93.5% of the sera of the susceptible children 28 days after vaccination, and the geometric mean antibody titre of these sera was low (1:6). Of the 80 initially seropositive children 21 (26.2%) showed a significant antibody response to the vaccine and this was influenced by the pre-existing antibody level. These data have further demonstrated the safety and efficacy of the live mumps vaccine in children.     

浓缩狂犬病疫苗接种后不良反应与血清抗体水平分析

报导了≥２．５ＩＵ浓缩狂苗人体接种后的不良反应其反应率为１．３１％与以往未浓缩常规苗的反应率为０．７３％比较两者具有显著性差异（Ｘ２＝６．９４,Ｐ＜０．０１）,浓缩苗以注射１～３针时的不良反应多,临床表现以局部极痛、红肿、瘙痒、急性荨麻疹和过敏性休克等。但其血清抗体阳转率７天时为４０．０％以上,１４天时为１００％,而常规苗仅是４０．０％,抗体滴度浓缩苗第７天时达到有效保护水平（中和试验）。显示了其抗体产生早,水平高特点,关于如何减少浓缩狂苗不良反应问题,作者提出了几点建议     

Experimental rabies in a great horned owl

A great horned owl (Bubo virginianus) was fed the carcass of an experimentally infected rabid skunk. The bird developed antibody titer to rabies, detected by passive haemagglutination, 27 days after oral inoculation by ingestion. The owl suppressed the infection until corticosteroid administration, after which a maximum antibody titer was attained. Evidence of active rabies viral infection was seen by fluorescent antibody staining of oral swabs, corneal impression smears and histologic tissue smears, by suckling mouse inoculation of oral swab washings, and by transmission electron microcopy. No clinical signs of rabies virus infection were observed.