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     

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。     

非典型肺炎灭活疫苗免疫动物效果评价

目的应用空斑减少中和试验对非典型肺炎灭活疫苗动物免疫血清进行中和抗体测定,以评价其免疫效果。方法用Vero-E6细胞接种6孔塑料细胞培养板,加两层含琼脂糖培养基,以中性红为染色剂建立空斑试验,以能减少50%的空斑为标准测定抗SARS中和抗体。结果三批非典型肺炎灭活疫苗免疫小鼠和家兔均产生较高的中和抗体。结论非典型肺炎灭活疫苗对两种动物具有良好的免疫效果。     

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…     

肠道病毒71型假病毒荧光定量检测方法在疫苗中和抗体检测中的应用

目的假病毒荧光定量方法（PVLA）应用于EV71疫苗动物样本和人体样本检测的适用性研究。方法分别采用细胞病变法（CPE）和PVLA方法检测82份EV71试验小鼠血清以及27份疫苗临床试验人体血清样本,比较两者检测的一致性。结果应用两种方法检测82份疫苗免疫小鼠血清,EV71中和抗体滴度的相关系数为0．842,Bland—Altman分析显示高度一致性;检测27份EV71临床试验人体血清样本的中和抗体,两种方法具有高度一致性。结论PVLA与CPE方法具有高度一致性,适用于疫苗免疫动物样本和临床评价人体样本EV71中和抗体的检测。     

不同ELISA检测狂犬病疫苗抗原性的比较

目前常用疫苗攻击试验(如小鼠效力试验)对人用和兽用狂犬病灭活疫苗进行效力检定,本文讨论了体外ELISA法代替体内效力试验的可行性.人用冻干狂犬病灭活疫苗选用原代狗肾细胞培养的PM株(PM-DKC)制备,兽用液体灭活疫苗分别用PV、SAD、PM和Flury-LEP株制备.采用两种免疫捕获ELISA(GPELISA 6-15和GP ELISA 78-9)及一种竞争ELISA(GP ELISA)检测疫苗糖蛋白(GP)抗原,这些试验均以生物素标记的GP-单克隆抗体(McAb)2-22为指示抗体;采用竞争     

中和抗体检测应用于新型冠状病毒mRNA疫苗效力分析

目的 通过对新型冠状病毒（2019 novel coronavirus,2019-nCoV）mRNA疫苗（简称新冠mRNA疫苗）免疫小鼠后产生的中和抗体进行检测，探索中和抗体检测法应用于mRNA疫苗体内效力评价的可行性。方法 采用6～8周BALB/c小鼠进行后肢肌肉免疫，检测不同免疫剂量和不同免疫程序的中和抗体滴度。并对8家企业生产的新冠mRNA疫苗免疫的小鼠血清进行中和抗体和IgG抗体检测。结果 2 µg、5 µg、10 µg不同剂量mRNA疫苗按照不同免疫程序免疫小鼠后中和抗体检测结果显示，抗体阳转率均为100%，中和抗体反应有明显的剂量-效应关系。2针间隔14 d加强免疫组抗体滴度显著高于间隔7d加强免疫组及一针组（F=57.13, P<0.001）。2 µg剂量间隔7 d加强免疫和间隔14 d加强免疫产生的中和抗体几何平均滴度（geometric mean titer,GMT）分别为218和468，差异有统计学意义（t=3.40， P=0.003）；5 µg剂量间隔7 d加强免疫和间隔14 d加强免疫产生的中和抗体GMT分别为499和1 436，差异有统计学意义（t=3.62，P=0.002）；10 µg剂量间隔7 d加强免疫和间隔14 d加强免疫产生的中和抗体GMT分别为608和1 909，差异有统计学意义（t=3.23，P=0.005）。国内8家企业生产的新冠mRNA疫苗免疫小鼠后均可产生高滴度的IgG抗体(104.5～107.5)和中和抗体(102.6～104.8)，效力测定结果均符合企业质量标准。各企业生产的mRNA疫苗的中和抗体滴度结果差异有统计学意义（F=70.03，P＜0.001）。结论 小鼠免疫后中和抗体检测可用于mRNA疫苗的体内效力评价。     

霍乱疫苗效力试验替代方法的研究

目的研究霍乱疫苗效力试验的替代方法。方法以不同剂量霍乱疫苗免疫小鼠,用间接ELISA方法检测小鼠全血的抗菌、抗毒抗体滴度,小鼠毒菌攻击法计算保护率。结果抗毒、抗菌抗体水平与免疫剂量和攻毒后的动物保护率成正相关,结果稳定。结论可以通过检测高免疫剂量组实验动物的抗菌、抗毒抗体替代以往小鼠攻毒法的效力试验。     

免疫吸附剂类型对狂犬病抗体酶免疫测定的影响:纯化的糖蛋白优于全病毒

近年来,酶免疫测定(EIA)已用于狂犬病病毒抗体的测定。为确定最适当的免疫吸附剂,作者用3种不同的体外法来测定人二倍体细胞(HDC)疫苗和神经组织[乳鼠脑(SMB)和羊脑(SB)]疫苗免疫的人和小鼠血清中的狂犬病病毒抗体:(1)用3种类型免疫吸附剂(完整狂犬病病毒、纯化的糖蛋白和纯化的核衣壳)作EIA;(2)快速荧光灶抑制试验(RFFIA)测定中和抗体;(3)快速的细胞内抗原酶免疫滴定法(REITICA)测定中和抗体。3种试验结果经最小平方直线回归分析进行比较。用标准离差法来比较相关系数。     

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。     

Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC₅₀ of 0.25 μM against cowpox and 0.8 μM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.     

Camelpox virus

Camelpox virus (CMLV) causes a smallpox-like illness in a unique host, the camel. The disease is enzootic in almost all regions where camel husbandry is practiced, and is responsible for severe economic losses. Although it is genetically the closest known virus to variola virus, the etiologic agent of smallpox, CMLV remains poorly studied. It is characterized by a narrow host range, the capacity to induce giant cells in culture and to counteract host immune defenses; however, the genetic bases associated with these features are not understood. Also, it still needs to be demonstrated whether CMLV strains of variable virulence circulate and how arthropod vectors might be involved in virus transmission. Current evidence indicates that, under certain circumstances, CMLV can be mildly pathogenic in humans. A reservoir host other than camels is unlikely to exist. We review here current knowledge of CMLV, including clinical and laboratory aspects of the disease. We also discuss prevention and therapy by use of vaccines and antiviral treatments, as well as the possibility of camelpox eradication.     

无小鼠神经毒力的乙型脑炎病毒/登革病毒1型嵌合病毒的筛选与序列分析

目的  采用蚀斑纯化法筛选对小鼠无神经毒力的乙型脑炎病毒（Japanese encephalitis virus,JEV）/登革病毒（dengue virus,DENV）1型嵌合病毒（JD1）纯化株,分析影响JD1小鼠神经毒力的相关位点。方法  将JD1在乳地鼠肾细胞中进行3轮蚀斑纯化,检测JD1纯化株对小鼠的脑内神经毒力。然后提取无神经毒力纯化株的基因组RNA,用逆转录PCR分段扩增全基因组序列并进行序列测定。将测序结果分别与原DENV-1 的前膜-包膜蛋白和JEV骨架病毒株SA-14-14-2的基因序列进行比对。选取2个无神经毒力纯化株进行小鼠免疫保护实验。采用单侧t检验对结果进行比较。结果  经过3轮蚀斑纯化后,获得大、中、小3种蚀斑形态的6个JD1纯化株。3个纯化株对小鼠无神经毒力,小鼠脑内注射后全部存活;1株神经毒力较弱,70%以上小鼠存活;2株具有强神经毒力,小鼠全部死亡。测序结果显示,对小鼠无神经毒力的3个纯化株存在2处相同的氨基酸突变。将其中2个纯化株JD1-PP-61和JD1-PP-31腹腔免疫小鼠后,再用1 000半数致死量DENV-1鼠脑适应株进行脑内攻击。JD1-PP-61能较好地保护小鼠抵抗病毒攻击,仅20.0%小鼠死亡（t=7.237,P<0.001）;JD1-PP-31保护效果稍弱,41.9 %小鼠死亡（t=4.752,P<0.01）。结论  通过蚀斑纯化筛选出对小鼠无神经毒力的JD1纯化株,并发现了可能与神经毒力减弱相关的位点。     

Notoginsenoside ST-4 inhibits virus penetration of herpes simplex virus in vitro

Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in raw and treated forms for a long time, led to the isolation of a new dammarane-type saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3β,12β,20(S)-tri-hydroxydammar-24-ene-3-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-β-d-glu-copyranoside, based on the detailed analyses of the 1D and 2D NMR spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50% effective concentration (EC₅₀) values, determined by plaque reduction assay, were 16.47 ± 0.67 and 19.44 ± 1.16 μM for HSV-1 and HSV-2, respectively, whereas the 50% cytotoxic concentration (CC₅₀) determined by the XTT test on Vero cells was 510.64 ± 4.56 μM. As analyzed by attachment assay and penetration assay based on plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due to the penetration inhibition effects, which was confirmed by fluorescence microscopy observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore, notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.     

Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference

Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR + U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.     

流感病毒裂解疫苗及单价病毒液的血凝素稳定性研究

目的 通过对连续三年生产的流行性感冒(流感)病毒裂解疫苗成品和单价病毒液(MVP)血凝素(HA)的定期检测,了解成品和MVP的HA含量的变化情况,以便更准确、有效地配制半成品.  方法 2003-2005年,每年选择一定数量的成品和MVP,按一定时间间隔抽样,采用单向免疫扩散法检测成品和MVP的HA含量.观察期至少为1年.  结果 成品存放至半年,HA含量都在控制范围之内.但存放至1年,个别毒株HA含量降至低于控制范围.MVP的HA稳定性因毒株的不同而异.   结论 一般情况下,流感疫苗在成品检定合格后7个月内完成使用,因此,在此期间,成品的HA含量在合格范围之内.不同毒株的MVP,HA降幅不同,可能是由于编码HA蛋白的RNA4节段的核苷酸序列在不同亚型,甚至在同一亚型不同变异株间的差异所造成的.