人用狂犬病灭活疫苗的临床研究和应用

狂犬病疫苗是预防狂犬病的安全有效方法。本文从人二倍体细胞疫苗、纯化Vero细胞狂犬病疫苗、层析纯化狂犬病疫苗的临床研究角度,概述此类广泛应用的人用狂犬病疫苗的优缺点、国内外的发展过程和临床免疫效果,以及我国当前对氢氧化铝佐剂疫苗和无佐剂疫苗的临床研究热点。数据显示国产无佐剂疫苗引起的不良反应发生率和抗体阳转率与进口同类疫苗相近,这类疫苗预防狂犬病的免疫效果是有保证的。     

狂犬病疫苗的干扰素原性与治疗和预防效果的关系

作者用无干扰素(IFN)原性的提纯的羊脑狂犬病疫苗和有IFN原性的日本鹌鹑胚细胞培养狂犬病疫苗进行了比较,以阐明IFN原性对预防和治疗效果的意义.小鼠经腹腔接种细胞培养疫苗后,4小时血清中可测出IFN;8～12小时达到高峰;24小时后不能测出.2周后再接种该疫苗未测出IFN.用提纯的羊脑疫苗免疫后.未观察到IFN原性.作者进一步比较了两种疫苗的治疗和预防效果.间隔两周免疫两次,7天后测定血清中和抗体滴度和对固定狂犬病病毒(CVS株)脑内感染的抵抗力,发现两者无明显差异.     

叙利亚地鼠作为狂犬病暴露后免疫动物模型的研究

目的  对叙利亚地鼠（地鼠）作为狂犬病暴露后疫苗免疫效果动物模型的可行性进行研究。方法 将狂犬病病毒CVS株和CTN-1V株分别肌内感染不同周龄的地鼠和小鼠,观察动物对不同毒株的敏感性。用直接免疫荧光法检测CVS株进入地鼠脑组织的情况;用快速荧光灶抑制试验检测血清中和抗体。以不同病毒量CVS株感染8周龄雌性地鼠,确定暴露时病毒的最佳感染剂量。对暴露后地鼠用疫苗进行免疫,观察疫苗的保护效果。结果  地鼠感染后7 d左右出现狂犬病临床症状,同一病毒株对地鼠的致病力比小鼠强〔6.4～8.0 lg半数致死量（LD₅₀）/ml对3.4~6.5 lgLD₅₀/ml〕;3周龄和8周龄地鼠对病毒的敏感性无差异。感染后5~6 d病毒进入地鼠脑组织。以3.8～4.0 lg小鼠脑内半数致死量（MICLD₅₀）/ml的CVS株感染后6～7 d,地鼠血清中检出中和抗体。暴露后疫苗免疫结果显示,不同疫苗具有不同程度的保护效果。结论  地鼠对狂犬病病毒神经外途径感染敏感,临床症状典型,潜伏期恒定,感染后免疫血清抗体应答出现早,具备作为暴露后疫苗免疫动物模型的条件。     

不同狂犬病疫苗初免和加强免疫后的中和抗体水平比较

作者对实验性狂犬病 DNA疫苗、编码狂犬病病毒糖蛋白基因的重组痘苗病毒疫苗(RVV)和市售人二倍体细胞病毒灭活疫苗(HDCV)分别免疫小鼠后的中和抗体持续时间及加强免疫后的回忆应答进行了比较。　　小鼠经 3种狂犬病疫苗初免后 90天所产生的中和抗体滴度均超过 0 .5 IU/ ml。RVV组的抗体几何平均滴度 (GMT)分别高于 HDCV和 DNA疫苗组 1 0倍和 1 0 0倍以上 ,初免后第 5 40天 ,RVV免疫鼠的中和抗体滴度高于 HDCV或 DNA疫苗免疫鼠 1 0倍以上。　　接受 3种狂犬病疫苗初免后的小鼠 ,再以 HDCV加强免疫后均能诱导早期 (7天 )稳定…     

狂犬病疫苗NIH效力试验体液中和抗体的保护作用

本文阐明了狂犬病疫苗腹腔免疫小鼠后,体液中狂犬病病毒中和抗体应答对保护作用的重要性。作者用7～9周龄的雌性ICR和Balb/C小鼠,按NIH试验要求进行免疫和攻击。狂犬病疫苗(SVR-TC)系Pasteur株在BHK组织培养中增殖所得。颅内攻击毒株为鼠脑悬液中的CVS-11,稀释到10～50半数致死量(LD_(50))。小鼠眼眶采血分离血清,用快速荧光灶抑制试验(RFFIT)测定病毒中和抗体(VNA)滴度,血清IgM中和抗体滴度经2-巯基乙醇变性后测定。     

不同毒株对狂犬病疫苗小鼠效力试验的影响

作者按欧洲药典标准效力试验方法,用狂犬病病毒CVS、SAD和LEP株制备的三种疫苗对小鼠进行腹腔免疫后,用三种疫苗病毒株和一种狐狂犬病野毒株(GS7)作交叉攻击试验.结果表明,各免疫组对GS7的攻击保护性最好;其次,对同源毒株的攻击保护性亦好.将上述三种疫苗5倍稀释后分别对小鼠进行腹腔免疫,14天后采血.用小鼠中和试验(MNT)或快速荧光灶抑制试验(RFFIT)测定抗体.发现CVS疫苗免疫动物所获得的抗血清对同源毒株的中和效价(ED_(50))较     

儿童接种狂犬病纯化疫苗的免疫效果和安全性观察

目的 观察狂犬病纯化疫苗在儿童中的免疫效果和安全性.方法 对注射狂犬病疫苗的337例儿童进行观察并应用酶联免疫吸附法进行血清抗体检测.结果 接种疫苗后阳性率达99.7%,不良反应轻微.结论 国产无佐剂液体疫苗、国产冻干疫苗及进口冻干疫苗对儿童均具有良好的免疫原性,阳性率满意,安全性好.     

RFFIT法血清系列参考品的建立

目的建立血清参考品,用于RFFIT法检测抗狂犬病毒中和抗体。方法狂犬病疫苗免疫后人血清,采用小鼠脑内中和试验确定抗狂犬病毒中和抗体滴度后,混匀,分装,以第2批人免疫球蛋白国际标准品为参考品用RFFIT方法进行标定,并考察所建参考品的稳定性。结果检测用标准血清效价为22.14IU.mL-1,弱阳性、强阳性参考品分别1.11IU.mL-1,9.20IU.mL-1,阴性参考品低于0.1IU.mL-1。结论建立的系列参考品适用于RFFIT法检测人血清中抗狂犬病中和抗体水平。     

人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性研究

 目的   比较人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性,为该疫苗的类型选择提供初步依据。 方法   采用相同血凝素含量（5 μg）的H7N9全病毒灭活和裂解疫苗（含或不含氢氧化铝佐剂共4种类型）,分别对BALB/c小鼠进行1针或2针免疫。免疫后,用血凝抑制（hemagglutination inhibition,HI）试验检测血清抗体滴度,比较不同类型疫苗的免疫效果。 结果   小鼠免疫1针全病毒灭活疫苗后,全部血清阳转,HI抗体几何平均滴度（geometric mean titer,GMT）为149;免疫2针后,抗体GMT为243。小鼠免疫1针裂解疫苗后无抗体阳转;免疫2针后全部抗体阳转,GMT为139。两种疫苗添加铝佐剂后,诱导的HI抗体GMT仅略有增加。 结论   H7N9全病毒灭活疫苗在小鼠中的免疫原性较强。在同样类型和剂量的情况下,裂解疫苗需要免疫两次才能达到与全病毒疫苗相同的效果。铝佐剂对免疫原性提升不明显。     

SARS灭活疫苗毒种特性和效力试验方法的研究和建立

目的 验证SARS灭活疫苗生产用毒种和建立疫苗效力试验方法和评价疫苗有效性的方法和初步标准。方法 将SARS灭活疫苗进行系列稀释后免疫小鼠、原液疫苗免疫家兔，用异株病毒作为攻击毒以蚀斑减少法测定血清中和抗体，并与一组SARS病人恢复期血清的中和抗体水平进行比较。初步建立疫苗的效力试验方法和评价标准。结果 疫苗以不同稀释度免疫小鼠，能产生随不同稀释度疫苗相应梯度的中和抗体滴度。用原倍疫苗免疫的小鼠血清中和抗体可达495．2；原倍疫苗免疫家兔所产生的中和抗体GNT为55．0～79．9。无论是小鼠或家兔所产生的中和抗体对广东分离的病毒均较北京分离的病毒为高。南北不同区域的SARS病人恢复期血清中和抗体GMT为50．12～54．95，且发现北方人群样本的抗体高于南方人群样本。结论 用小鼠和家兔作疫苗的效力试验模型具有较好的敏感性和重复性，且产生的抗体水平较高：选用的攻击病毒对多批用不同毒株生产的疫苗免疫后的动物抗体具有较高的一致性，可用于评价疫苗的效力。     

Posintro™-HBsAg, a modified ISCOM including HBsAg, induces strong cellular and humoral responses

To improve the hepatitis B vaccines on the market new adjuvant systems have to substitute aluminium. In this study the hepatitis B surface antigen (HBsAg) was incorporated into a novel adjuvant system, the Posintro?, a modification of the traditional immune stimulatory complexes (ISCOMs). This new HBsAg vaccine formulation, Posintro?-HBsAg, was compared to two commercial hepatitis B vaccines including aluminium or monophosphoryl lipid A (MPL) and the two adjuvant systems MF59 and QS21 in their efficiency to prime both cellular and humoral immune responses. The Posintro?-HBsAg induced the strongest humoral response with high titers of HBsAg specific antibody, high number of antigen specific B-cells and a strong T helper 1 (Th1) antibody profile when compared to the other adjuvant formulations. The Posintro?-HBsAg was also a strong inducer of cellular immune responses with induction of delayed type hypersensitivity (DTH) reaction and CD4(+) T-cell proliferation. In addition, Posintro?-HBsAg was the only vaccine tested that also induced a strong cytotoxic T lymphocyte (CTL) response, with high levels of antigen specific CD8 T-cells secreting IFN-gamma mediating cytolytic activity. The results demonstrate that this novel experimental vaccine formulation, the Posintro?-HBsAg, is strongly immunogenic and can induce both class I and class II responses in experimental animals. This shows promise both for the protection against hepatitis B virus infection and as a potential therapeutic vaccine.     

Comparison of the adjuvanticity of two adjuvant formulations containing de-<Emphasis Type="Italic">O</Emphasis>-acylated lipooligosaccharide on Japanese encephalitis vaccine in mice

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT₅₀) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.     

Influenza vaccine and adjuvant

Adjuvant is originated from the Latin word "adjuvare" which means "help" in English to enhance the immunological responses when given together with antigens. The beginning of adjuvant was mineral oil which enhanced the immune response when it was given with inactivated Salmonella typhimurium. Aluminium salt was used to precipitate diphtheria toxoid and increased level of antibody response was demonstrated when administered with alum-precipitated antigens. Since 1930, aluminium salt has been used as DTaP (diphtheria-tetanus-acellular pertussis vaccine) adjuvant. Many candidates were tested for adjuvant activity but only aluminum salt is allowed to use for human vaccines. New adjuvant MF59, oil-in-water emulsion type, was developed for influenza vaccine for elderly (Fluad) and series of AS adjuvant are used for hepatitis B, pandemic flue, and human papiloma virus vaccines. Oil-adjuvanted influenza pandemic vaccines induced higher antibody response than alum-adjuvanted vaccine with higher incidence of adverse events, especially for local reactions. Alum-adjuvanted whole virion inactivated H5N1 vaccine was developed in Japan, and it induced relatively well immune responses in adults. When it applied for children, febrile reaction was noted in approximately 60% of the subjects, with higher antibodies. Recent investigation on innate immunity demonstrates that adjuvant activity is initiated from the stimulation on innate immunity and/or inflammasome, resulting in cytokine induction and antigen uptake by monocytes and macrophages. The probable reason for high incidence of febrile reaction should be investigated to develop a safe and effective influenza vaccine.     

Lyophilized Filovirus Glycoprotein Vaccines: Peroxides in a Vaccine Formulation with Polysorbate 80–Containing Adjuvant are Associated with Reduced Neutralizing Antibody Titers in Both Mice and Non-Human Primates

Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus are the filoviruses most commonly associated with human disease. Previously, we administered a three-dose regimen of trivalent vaccines comprising glycoprotein antigens from each virus in mice and non-human primates (NHPs). The vaccines, which contained a polysorbate 80-stabilized squalane-in-water emulsion adjuvant and were lyophilized from a solution containing trehalose, produced high antibody levels against all three filovirus antigens. Subsequently, single-vial formulations containing a higher concentration of adjuvant were generated for testing in NHPs, but these vaccines elicited lower neutralizing antibody titers in NHPs than previously tested formulations. In order to explain these results, in the current work we measured the size of adjuvant emulsion droplets and the peroxide levels present in the vaccines after lyophilization and reconstitution and tested the effects of these variables on the immune response in mice. Increases in squalane droplet sizes were observed when the ratio of adjuvant to trehalose was increased beyond a critical value, but antibody and neutralizing antibody titers in mice were independent of the droplet size. Higher levels of peroxides in the vaccines correlated with higher concentrations of adjuvant in the formulations, and higher peroxide levels were associated with increased levels of oxidative damage to glycoprotein antigens. Neutralizing titers in mice were inversely correlated with peroxide levels in the vaccines, but peroxide levels could be reduced by adding free methionine, resulting in retention of high neutralizing antibody titers. Overall, the results suggest that oxidation of glycoprotein antigens by peroxides in the polysorbate 80-stabilized squalane-in-water emulsion adjuvant, but not lyophilization-induced increases in adjuvant emulsion droplet size may have been responsible for the decreased neutralizing titers seen in formulations containing higher amounts of adjuvant.     

Water-in-oil solubilized vaccine adjuvants

The solubilization of water and toxoid solutions in oils by non-ionic surface-active agents has been examined. Water-in-oil solubilized adjuvant formulations of vaccines containing Clostridium welchii type D toxoid as antigen have been tested in laboratory animals. The antitoxin titres in rabbit serum induced by the most successful formulation were as high 14 weeks after a single dose, as the peak titres after two doses of a simple aluminium hydroxide adsorbed vaccine. The vaccines are clear and of low viscosity which facilitates accurate measurement and handling by syringe.     

双抗夹心ELISA法快速检测人用狂犬病疫苗的效价

目的：用单克隆抗体制备的双抗夹心ELISA法，快速检测人用狂犬病疫苗中的狂犬病毒糖蛋白G的含量，并探讨其替代NIH法的可行性。方法：将疫苗样品用NIH法检定，同时以国家标准品为参考疫苗，用双抗夹心ELISA法检测。结果：用双抗夹心ELISA法测得的IgED50为2．60～2．78；用NIH法测得的IgED50为2．55～2．85。对两种检测方法进行F检验，两种检测方法的灵敏度有显著差异（F=5．76，P〈0．05）；用t检验法对这两种检测方法进行分析，表明两组测定值的差异无统计学意义（t=0．187，P〉0．05）。结论：双抗夹心ELISA法与NIH法测得的IgED50呈正相关性。与NIH法相比，双抗夹心ELISA法具有重复性好、成本低、快速等优点。用双抗夹心ELISA法取代NIH法测狂犬病疫苗效价是可行的。     

Immunogenicity of a scalable inactivated rotavirus vaccine in mice

Inactivated rotavirus vaccine is a safe and effective potential vaccine for the prevention of rotavirus infection among children, but no approved licensed vaccine is available now. In this study, a scalable inactivated rotavirus vaccine, prepared in Vero cells cultured by microcarrier fermentation, inactivated by formalin and absorbed by Al(OH)(3) adjuvant, was vaccinated into the six weeks-old female Balb/c mice by intramuscular injection. After twice immunization at interval of three weeks, both humoral and cell-mediated immune responses were assessed by ELISA, microneutralization assay and EISPOT assay. The results indicated that the scalable inactivated rotavirus vaccines induced not only high serum IgG antibody and neutralizing antibody responses, but Th1 and Th2 cytokine-secreting cell responses in mice immunized by the inactivated rotavirus vaccines. These results suggest that the scalable inactivated rotavirus vaccine has good immunogenicity, which provided the base for the scaled development of inactivated rotavirus vaccine in the future.     

禽流感H5N1灭活疫苗与不同纳米化佐剂联合免疫研究

目的 观察禽流感H5N1灭活疫苗加不同佐剂以及纳米化佐剂免疫小鼠后产生的免疫应答的差异,同时观察免疫对异亚型病毒攻击后的保护情况.   方法 用H5N1灭活疫苗分别联合佐剂氢氧化铝、纳米化氢氧化铝、MF59和纳米化MF59通过腹腔注射方式免疫雌性BALB/c小鼠,同时分别以H5N1灭活疫苗免疫小鼠以及PBS腹腔注射处理小鼠作对照.采用ELISA方法分别对各组小鼠免疫后血清特异性IgG及其亚类IgG1、IgG2a水平进行检测,以PR8病毒鼻腔攻击后观察小鼠体重变化情况和生存率.采用t检验作组间比较.    结果 与PBS处理组相比,无论以何种方式免疫H5N1疫苗,免疫后特异性IgG及其亚类水平均明显升高(t=7.4004,P<0.01),以联合MF59后诱导的特异性抗体水平最高.其中疫苗单独免疫组和联合M59免疫组IgG2a水平升高明显,联合纳米化MF59免疫小鼠后IgG2a水平有所下降,IgG1水平有所升高;联合铝佐剂组以IgG1升高为主.攻毒后各组小鼠体重均出现下降,但疫苗单独免疫小鼠以及疫苗与佐剂联合免疫小鼠于攻毒后期体重恢复接近正常或者正常.PBS处理组小鼠攻毒后全部死亡,佐剂联合免疫组小鼠存活率100%,而疫苗单独免疫小鼠存活率为70%.   结论 H5N1疫苗无论是单独免疫或是联合不同佐剂免疫均可诱导较高水平的特异性抗体产生,有非常好的免疫原性.H5N1灭活疫苗诱导的抗体亚类以IgG2a为主,MF59以及纳米化MF59也以诱导IgG2a亚类为主,但纳米化MF59可诱导更均衡的免疫应答.联合铝佐剂或纳米化铝佐剂免疫小鼠后均可诱导以IgG1亚类为主的抗体应答.联合两种佐剂均可对小鼠产生很好的异亚型保护.     

Immunogenicity study of abbreviated rabies preexposure vaccination schedules

OBJECTIVE: To evaluate abbreviated preexposure rabies vaccination schedules that would reduce cost and shorten time required for completion. METHOD: A random prospective immunogenicity study, using a group of 96 volunteer preclinical veterinary students, primary school children, and hospital-based health care workers. They were divided into six groups and administered abbreviated schedules of preexposure tissue culture rabies vaccines. Neutralizing antibodies were determined on days 0 and 360, and following boosters on days 367 and 374. RESULTS: All subjects, including one group that received only 0.1 mL intradermally at two sites on one day, had detectable neutralizing antibody titers 1 year later and responded with an accelerated antibody response when given booster injections. CONCLUSION: It might be possible to develop a 1-week and even one clinic visit preexposure vaccine schedule that would provide at least 1 year of immune memory.