流感病毒中和性基因工程Fab抗体的研制

目的 获得基因工程抗流感病毒抗体,为检测其粘膜用药在动物模型中的抗病毒效果奠定基因。方法 从酶联免疫吸附试验阳性的人外周血中分离淋巴细胞,提取总RNA,Oligo-dT逆转录cDNA,聚合酶链反应扩增人IgG轻、重链基因,利用pComb3载体构建噬菌体抗体库。用纯化的流感病毒A／Sydeny/5/97(H3N2）为固相抗原筛选抗体Fab段,并在大肠埃希菌中进行分泌性表达。通过血凝抑制实验、免疫荧光实验和病毒中和实验选择具有中和作用的Fab抗体。结果 分离到两株Fab克隆,IV－2和IV－6,流感病毒抗原和抗Fab抗体直接ELISA检测阳性,间接免疫荧光实验呈阳性,它们都具有血浆抑制作用,病毒中和实验显示能使病毒滴度下降30倍和20倍。结论 获得了两株具有病毒中和活性的人源抗流感病毒悉尼株的Fab段抗体,为进一步的表达纯化及粘膜给药研究其抗病毒效果提供材料。     

疫苗中和抗体体外检测方法的研究进展

中和抗体是抗病毒免疫的主要因素，也是疫苗免疫治疗效果的重要检测指标之一。预防疫苗的免疫保护性主要体现于是否可诱导出高滴度的中和抗体，因此中和抗体活性也是疫苗评估和质控的重要依据。迄今为止，根据已知抗原特性、抗原与抗体反应特点以及结果判定方法的不同，已建立了多种病毒中和抗体活性的体外测定方法。本文就近年来关于中和抗体活性体外测定方法的最新研究进展做一综述。     

灭活SARS病毒的免疫原性的实验研究

研究灭活SARS病毒的免疫原性。SARS病毒F6 9株经甲醛灭活后 ,加入氢氧化铝佐剂 ,以 3种剂量接种BALB/c小鼠 ,定时采血 ,测定特异性抗体的滴度及其中和活性 ,同时用化学发光酶联免疫法测定抗血清与SARS病毒结构蛋白的反应特异性及其相对强度。结果发现 ,小鼠接种疫苗 4d后 ,血清中可检测到IgM抗体 ,直至 2 6d后逐渐下降 ;IgG抗体在初次免疫 8d后出现 ,4 7d时达最高峰 ,6 3d后进入稳定期 ;不同剂量组的抗体滴度具有明显剂效关系 ,低剂量组和中剂量组滴度峰值为 1∶192 0 0 ,高剂量组滴度峰值为 1∶384 0 0。中和实验结果表明 ,小鼠所产生的抗体具有中和病毒活性 ,在 6 3d时 ,低剂量组和中剂量组血清的中和效价为 1∶12 80 ,高剂量组血清的中和效价为 1∶5 12 0。抗体分类结果表明 ,小鼠抗血清中含有针对多种SARS病毒结构蛋白的特异性抗体 ,其中 ,针对N蛋白、S4蛋白和S2蛋白的抗体水平相对较高 ,而抗M抗体、抗 3CL抗体的水平相对较低。上述结果说明 ,SARS病毒F6 9株经甲醛灭活后 ,各主要结构蛋白仍保持较强免疫原性 ;免疫小鼠后 ,可以诱导产生高滴度的特异性混合抗体 ,在体外可以保护敏感细胞不受SARS病毒攻击     

汉滩病毒感染成龄鼠模型用于特异性单克隆抗体保护作用的研究

目的建立汉滩病毒感染成龄鼠的模型,以进行mAb的保护实验.方法将7～8wkBalb/c小鼠于感染病毒前1d及感染后1d,2d和4d,分别经腹腔注射环磷酰胺65mg/kg体重,总剂量为260mg/kg体重,观察其发病及死亡情况,并检测其体内不同脏器中汉滩病毒抗原的分布及滴度.用mAb对上述感染小鼠进行保护实验.结果经环磷酰胺处理的成龄鼠,由汉滩病毒隐性感染变为急性致死性感染(死亡率达100%).其发病症状与汉滩病毒感染的乳鼠类似,平均发病与死亡时间较感染乳鼠晚2～3d,且较为规律和集中.汉滩病毒抗原在感染的成龄鼠和乳鼠体内的分布及滴度基本相同.用mAb对感染的成龄鼠和乳鼠分别进行保护实验,结果完全一致.结论经环磷酰胺处理的成龄鼠,可作为汉滩病毒和HFRS研究用的动物模型.     

大黄乙醇提取物体内抗单纯疱疹病毒作用的研究

目的　了解大黄乙醇提取物 (大黄 )体内抗单纯疱疹病毒 (HSV)的作用。方法　小鼠尾静脉接种滴度为 10 3TCID50 HSV Ⅰ型 0 15ml,第 2天皮下注射给药。将BALB c小鼠分为 7个组 ,分别给予不同剂量的药物 ,不同时间取肝、脾、肾等组织 ,做病理切片 ,观察病变情况 ;用空斑形成法滴定各脏器中的病毒滴度 ,观察药物在动物体内对HSV感染的抑制作用。结果　大黄乙醇提取物经皮下注射未发现小鼠有毒性反应 ;大黄各治疗组脾脏不出现病理变化 ,中、高剂量组肝脏、肾脏的病理损伤也能逐渐消失 ;大黄低剂量组与ACV阳性对照组效果相当 ;病毒滴度测定说明大黄能使各脏器中病毒滴度迅速下降 ,中、高剂量组比低剂量组作用明显 ;将各组数值进行两两比较 (Q检验 ) :F =4 9 14 5 9,P <0 0 0 1,总体均数差异有显著性 ;治疗组 (ACV、DH1、DH2、DH3)与非治疗组 (VC)差异有显著性 (P <0 0 1) ;DH2、DH3、与DH1差异有显著性 (P <0 0 1) ;DH1、DH2、DH3与ACV差异无显著性 (P >0 0 5 )。结果说明 ,对于总滴度平均值的下降 ,大黄与ACV同样有效 ,且中、高剂量组的效果优于低剂量组。结论　大黄乙醇提取物在体内具有非常明显的抗HSV感染作用 ,应用前景广阔。     

人乳头瘤病毒(HPV)L2肽免疫小鼠诱导的抗体水平与保护作用的关系研究

目的 检测HPV-58 L2 11～200 AA肽在动物体内对HPV的保护效果,并分析疫苗诱导的抗体滴度或中和抗体滴度与疫苗保护作用之间的对应关系.方法 利用大肠杆菌表达HPV-58L2 11～200 AA肽,纯化目的 蛋白并与铝佐剂吸附后免疫小鼠,利用小鼠HPV-58假病毒感染模型检测不同剂量的免疫原对小鼠的保护作用.通过ELISA和假病毒中和抗体检测方法 检测免疫血清中的总抗体水平和中和抗体水平,分析具有保护作用的抗体或中和抗体滴度.结果 当蛋白免疫剂量为8μg时,能够完全保护小鼠不受HPV假病毒的感染.小鼠免疫血清中的中和抗体水平较低,利用已建立的假病毒中和试验方法 检测不到血清中的中和抗体.而利用ELISA检测血清中的总抗体水平结果 显示,免疫血清中的总抗体水平与疫苗的保护效果之间存在对应关系,当抗体滴度大于等于1:25 000时,小鼠体内检测不到荧光信号,能够保护机体不受假病毒的感染.结论 HPV-58 L2 11～200 AA肽能够保护小鼠不受假病毒的感染,L2 11～200 AA肽具有较好的疫苗发展前景,其引发的总抗体水平可以作为评价保护效果的间接指标.     

加巴喷丁体内外抗RSV感染的作用

目的探讨加巴喷丁对呼吸道合胞病毒(respiratory syncytial virus,RSV)的抗病毒作用。方法体外实验通过MTS、TCID50、q RT-PCR方法检测不同浓度加巴喷丁对RSV感染的抑制作用,包括细胞活力、病毒复制及细胞因子的变化。体内实验选取4~6周龄C57BL/6小鼠,随机分为空白对照组、RSV感染组、低剂量及高剂量加巴喷丁处理组,连续腹腔注射给药,每日观察体质量变化,HE染色观察小鼠肺部的病理变化,q RT-PCR方法检测肺部病毒载量。结果 1、2、5、10 mmol/L不同浓度加巴喷丁显著增加RSV感染A549细胞的活力;5、10 mmol/L浓度的加巴喷丁可显著降低RSV感染A549细胞的病毒载量,10 mmol/L的加巴喷丁可显著抑制病毒的复制,减少CCL3、CCL5、CXCL2及TNF-α、IL-6、IL-8等趋化因子和炎性因子表达,促进干扰素IFN-α、IFN-β表达;动物实验表明90μg、180μg的加巴喷丁处理组可减轻RSV感染小鼠的体质量变化、改善肺部病理损伤和降低病毒载量。结论加巴喷丁可通过抑制病毒复制,调节趋化因子及炎性因子释放,促进干扰素分泌的方式发挥体内抗病毒作用,对RSV感染的C57BL/6小鼠有一定的治疗作用,可改善肺部病理,为进一步临床应用提供实验依据。     

水痘带状疱疹病毒gE蛋白联合不同佐剂对小鼠免疫效果的比较

目的比较利用AS01和Al/CpG联合佐剂, 水痘带状疱疹病毒糖蛋白E(Varicella Zoster virus glycoprotein E, VZV gE)在BALB/c小鼠体内的免疫效果。方法 BALB/c小鼠分别于0和21天免疫, 小鼠血清抗体检测采用酶联免疫吸附实验;用VZV检测小鼠血清中和抗体滴度;酶联免疫吸附斑点实验检测细胞免疫反应。结果经过2次肌肉注射免疫, 实验组(Shingrix、gE+Al/CpG和gE+AS01)中小鼠均能产生高滴度的中和抗体, 增加分泌IFN-γ和IL-4的淋巴细胞数量。gE+Al/CpG免疫组中小鼠产生的中和抗体滴度最高(1943), 但是AS01佐剂组(Shingrix和gE+AS01)中小鼠产生的分泌IFN-γ和IL-4的淋巴细胞数量高于Al/CpG佐剂组(gE+Al/CpG)。相比于AS01佐剂, Al/CpG佐剂诱导小鼠产生了偏向Th2型的体液免疫应答。各实验组中小鼠CD4+T细胞、CD8+T细胞的比例均没有统计学差异。结论 Al/CpG佐剂联合gE蛋白诱导高滴度中和抗体, 但是诱导产生的细胞免疫应答强度低于AS01佐剂组。     

黄藤素注射液Beagle犬静脉给药毒性观察

目的：采用Beagle犬一次静脉滴注黄藤素注射液．观察其对试验动物所产生的急性毒性反应和死亡情况．为黄藤素注射液临床用药途径提供参考。方法：用近似致死剂量法(ALD)，选择3只健康6月龄Beagle犬，根据黄藤素注射液小鼠静脉注射给药急性毒性试验结果．按50％剂量递增．计算出剂量递增序列，在剂量序列范围内间隔一个剂最给一只动物。1号动物25．0mg/kg体重．2号动物50．0mg/kg体重．3号动物100．0mg/kg体重，     

新型抗蝰蛇毒血清对不同喹蛇毒作用的实验研究

目的　探讨新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的作用 .方法　采用免疫电泳、SDS -聚丙烯酰胺电泳及动物实验等方法 .结果　国产圆斑蝰蛇毒的LD50 (腹腔注射 )为 0 .3 0 6± 0 .0 0 3mg/kg ,缅甸蝰蛇毒的LD50 (腹腔注射 )为 0 .2 90± 0 .0 0 3mg/kg .免疫电泳结果表明国产圆斑蝰蛇毒和缅甸蝰蛇毒均和新型抗蝰蛇毒血清产生明显的免疫沉淀线 .体外中和实验表明抗蝰蛇毒血清对国产圆斑蝰蛇毒的中和抗体效价为 2 75 0 μg/ml(约45 0LD50 ) ,对缅甸蝰蛇毒的中和抗体效价为 2 490 μg/ml(约 43 0LD50 ) .体内保护实验表明给予 0 .0 5ml抗蝰蛇毒血清可抵御 2 5 4μg(约41 5LD50 )国产圆斑蝰蛇毒或 116μg(约 2 0LD50 )缅甸蝰蛇毒的攻击 ,使小鼠全部存活 .结论　新型抗蝰蛇毒血清对国产圆斑蝰蛇毒及缅甸蝰蛇毒的中和抗体效价均较高 ,有较大的应用价值 .     

Anti-influenza-virus activity of total alkaloids from <Emphasis Type="Italic">Commelina communis</Emphasis> L.

The antiviral activity of total alkaloids from Commelina communis L. (TAC) against influenza virus A/PR/8/34 (H1N1) was investigated in vitro and in vivo. TAC exhibited an inhibitory action on the growth of influenza virus in Madin-Darby canine kidney cells when added before or after viral infection. In mice infected with influenza virus, orally administered TAC at 8, 16 or 32 mg/kg per day for 6 days significantly increased the survival rate, prolonged the mean survival time and reduced the viral titers in the lung and the lung index, compared with that of the untreated virus control. The results obtained suggest that TAC has a pronounced protective effect against infection by influenza A virus.     

Antiviral activity of arbidol against influenza A virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo

Summary Arbidol, ethyl-6-bromo-4-[(dimethylamino)-methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-in dole-3-carboxylate hydrochloride monohydrate, is an antiviral chemical agent. In this report, we studied the antiviral activity of arbidol against a panel of human respiratory viruses, namely influenza A virus (FLU-A, A/PR/8/34 H1N1), respiratory syncytial virus (RSV), human rhinovirus type 14 (HRV 14), coxsackie virus B3 (CVB3) and adenovirus type 7 (AdV-7) in vitro in cell culture. Arbidol was found to present potent inhibitory activity against enveloped and non-enveloped RNA viruses, including FLU-A, RSV, HRV 14 and CVB3 when added before, during, or after viral infection, with 50% inhibitory concentration (IC₅₀) ranging from 2.7 to 13.8 μg/ml. However, arbidol showed selective antiviral activity against AdV-7, a DNA virus, only when added after infection (therapeutic index (TI) = 5.5). Orally administered arbidol at 50 or 100 mg/kg/day beginning 24 h pre-virus exposure for 6 days significantly reduced mean pulmonary virus yields and the rate of mortality in mice infected with FLU-A (A/PR/8/34 H1N1). Our results suggest that arbidol has the ability to elicit protective broad-spectrum antiviral activity against a number of human pathogenic respiratory viruses.     

The anti-tumor agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), induces IFN-beta-mediated antiviral activity in vitro and in vivo

The 2009 outbreak of pandemic H1N1 influenza, increased drug resistance, and the significant delay in obtaining adequate numbers of vaccine doses have heightened awareness of the need to develop new antiviral drugs that can be used prophylactically or therapeutically. Previously, we showed that the experimental anti-tumor drug DMXAA potently induced IFN-β but relatively low TNF-α expression in vitro. This study confirms these findings in vivo and demonstrates further that DMXAA induces potent antiviral activity in vitro and in vivo. In vitro, DMXAA protected RAW 264.7 macrophage-like cells from VSV-induced cytotoxicity and moreover, inhibited replication of influenza, including the Tamiflu?-resistant H1N1 influenza A/Br strain, in MDCK cells. In vivo, DMXAA protected WT C57BL/6J but not IFN-β(-/-) mice from lethality induced by the mouse-adapted H1N1 PR8 influenza strain when administered before or after infection. Protection was accompanied by mitigation of weight loss, increased IFN-β mRNA and protein levels in the lung, and significant inhibition of viral replication in vivo early after DMXAA treatment. Collectively, this study provides data to support the use of DMXAA as a novel antiviral agent.     

Inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza

In vitro, nitric oxide (NO) has been shown to have antimicrobial activity against a wide range of viruses, including influenza A virus. Therefore, we hypothesized that inhaled nitric oxide (iNO) would increase survival in vivo by reducing the viral load in C57Bl/6 mice infected with a lethal dose of influenza A/WSN/33 (H1N1; WSN/33) virus. NO was delivered to influenza-infected mice either continuously or intermittently at 80 or 160 ppm, respectively, using both prophylactic and post-infection treatment strategies. Murine survival and weight loss were assessed, and lung viral load was quantified via plaque assay. Here, we report that iNO administered prophylactically or post-influenza infection failed to improve survival of infected mice. No difference in lung viral load was observed between experimental groups. Although NO has antiviral activity against influenza A virus in vitro, iNO therapy provided no apparent benefit when used for treatment of influenza A virus infection in vivo.     

Utilization of the embryonated egg for in vivo evaluation of the anti-influenza virus activity of neuraminidase inhibitors

Previous studies have shown that embryonated egg provides a convenient and easy to use system for in vivo screening of anti-influenza virus inhibitors. However, it is not known whether this model is suitable for testing neuraminidase (NA) inhibitors, too. Therefore, the present study describes the evaluation of the ion-channel blockers amantadine and rimantadine in comparison with the NA inhibitors oseltamivir and zanamivir by using the influenza A virus hen’s egg model. The treatment was started immediately before or after the challenge dose was placed on the chorioallantoic membrane (CAM). Differences between the survival rate of treated and untreated chick embryos infected with influenza A virus were analyzed statistically. As result, the survival rate of chick embryos could be significantly increased when the treatment with amantadine, rimantadine, oseltamivir, or zanamivir was started before the CAM was inoculated with one egg infective dose 50% (EID₅₀) influenza A virus. When the drugs were administered shortly after viral inoculation, significant antiviral efficacy was shown for rimantadine, oseltamivir, and zanamivir. Antiviral efficacy could be demonstrated exclusively for both oseltamivir and zanamivir after the embryos were infected with higher challenge doses of 10² EID₅₀influenza A virus. In conclusion, the NA inhibitors oseltamivir and zanamivir have a significantly better antiviral activity against influenza A virus than amantadine and rimantadine tested in embryonated hen’s eggs. Therefore, this model can be a valuable alternative approach for in vivo pre-testing anti-influenza virus activity of NA inhibitors.     

Inhibition of influenza virus replication by cocaine

Cocaine has been shown to have a number of diverse effects on the immune system. The current investigators have previously demonstrated an inhibitory effect of cocaine on murine hepatitis virus replication in peritoneal macrophages in vitro. The present study was undertaken to examine the effects of cocaine on influenza virus replication and to further characterize that effect in an animal model. Cocaine was capable of inducing a dose-dependent reduction in influenza PR-8 replication using MDCK cells in vitro. Concentrations of 100 microg/ml caused a 50% reduction of virus. To further characterize the effect in vivo, C57Bl/6 mice infected with influenza PR-8 by intranasal instillation were given daily ip injections of 10 mg/kg cocaine just prior to and for 4 days after exposure to influenza. Lungs from mice exposed to cocaine had viral titers that were reduced approximately 50% compared to controls as demonstrated by hemagglutination titers. Additional studies suggest that this reduction appears to be caused by an increase of cocaine-induced interferon.     

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.     

Enhancement of host resistance against virus infections by MTP-PE, a synthetic lipophilic muramyl peptide--I. Increased survival in mice and guinea pigs after single drug administration prior to infection, and the effect of MTP-PE on interferon levels in sera and lungs

Muramyl tripeptide-phosphatidylethanolamine (MTP-PE, CGP 19835) displays prophylactic antiviral activity in mice infected with influenza viruses A and B, parainfluenza 1 virus or herpes simplex type 1 viruses (HSV/1) and in guinea pigs infected with herpes simplex type 2 viruses (HSV/2). MTP-PE is effective when given in a single intranasal dose as early as 1-4 weeks before infection. In the case of HSV/2 infections, prophylactic effectiveness can be demonstrated after a single topical application into the vagina seven days before infection. Antiviral effects are observed in response to doses as little as 0.001 mg/kg bodyweight. The activity of the substance seems to be inversely related to the size of the viral inoculum, but poor dose-effect relation is demonstrable in a dose-range extending over four to five orders of magnitude. Furthermore, the compound is devoid of antiviral effects in vitro. MTP-PE does not induce interferon (IFN) in serum and lung, nor does it influence kinetics or quantity of serum and lung IFN content in the course of viral infections. However, when given intranasally 7 days before an oral dose of tilorone, increased levels of IFN in lung suspensions are observed.     

Induction of antibody response by DNA immunization of newborn baboons against influenza virus.

Previous studies showed that DNA immunization of newborn mice with plasmids expressing influenza virus antigens induced protective immunity. We have now extended the study of neonatal responsiveness to DNA vaccines to nonhuman primates. Baboons immunized as neonates with plasmids expressing type A influenza virus hemagglutinin (HA) and nucleoprotein (NP) in doses ranging from 40 microg to 1 mg per plasmid per dose developed virus-specific humoral responses. The titer and kinetics of appearance of virus-specific IgG antibodies were dose dependent. Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as early as 1 month after birth in baboons immunized with the highest and intermediate doses of vaccine. Virus-neutralizing antibodies were detected in the group of baboons immunized with the highest dose. The specificity of virus-neutralizing antibodies was found to be directed against homologous determinants of HA; however, the IgG antibodies also cross-reacted with HA of a drift variant. Thus, DNA vaccination of newborn baboons with a prototype vaccine against influenza virus resulted in induction of specific humoral immunity.