靶向HIV和AIV包膜蛋白跨膜亚基的病毒进入抑制剂研究
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摘要
研究背景
艾滋病和禽流感成为当今中国乃至全世界有深远影响的重大流行疾病,是当前人类健康面临的重大威胁,但一直以来缺乏有效的治疗药物和疫苗。与SARS冠状病毒(SARS-CoV)相似,人类免疫缺陷病毒(HIV)和禽流感病毒(AIV)均为Ⅰ型包膜病毒,采用相似的病毒-宿主细胞膜融合机制,即病毒表面糖蛋白结合到宿主细胞受体后,启动病毒融合蛋白的一系列构象变化。包膜病毒进入靶细胞的过程需要病毒膜糖蛋白的介导,这一类型病毒的包膜蛋白一般都具有两个亚基,其中不跨膜的亚基(如HIV的gp120、AIV的HA1等)具有与靶细胞膜上病毒受体相结合的位点,而跨膜亚基(如HIV的gp41、AIV的HA2等)则介导病毒膜与靶细胞膜的融合。以上述研究为基础设计的病毒进入抑制剂,可以在病毒包膜糖蛋白中间体构象形成的短时间内,高效、特异地竞争结合其配体,从而阻止包膜糖蛋白的进一步折叠,达到抑制病毒入侵的目的,为病毒疾病的防治提供了新思路和策略。
虽然流感病毒也是Ⅰ型包膜病毒,但衍生自HA2的多肽不具有抑制AIV的活性,其原因可能与流感病毒本身的融合机制有关。流感病毒与靶细胞的融合发生在胞内体膜上,需要酸性环境的诱导。而衍生于包膜病毒的多肽,一般都要30个左右氨基酸残基的多肽才有效。多肽分子量大,很难通过细胞膜进入到胞内体与流感病毒HA2的相应区域结合,另外,即使进到胞内体中去了,pH为5左右的酸性环境对多肽的活性可能也具有破坏作用。相比之下,许多小分子化合物能够轻易地穿过靶细胞膜并进入胞内体,若小分子化合物能阻止流感病毒HA2构象的改变,就有可能具有抗流感病毒感染的作用。
HIV是一种感染人类免疫系统细胞的慢病毒,主要感染CD4~+T淋巴细胞,特征性的引起CD4~+T细胞逐步减少和进行性免疫缺陷,最后导致艾滋病(获得性免疫缺陷综合征),引起机会性感染并最终导致死亡。病毒与宿主免疫细胞的相互作用是影响疾病发展的重要因素。在HIV感染导致的AIDS疾病进展过程中,T细胞活化是一把双刃剑。一方面CD4~+T细胞的活化在清除病毒过程是非常必要的,成熟的CD4~+Th细胞是抗病毒免疫的关键因素。另一方面,HIV膜蛋白(Env)能结合CD4~+受体,在CD4~+T细胞中复制,由于病毒颗粒的持续表达,HIV感染导致T细胞增殖增加,T细胞活化后又可借助自身表达的凋亡受体与配体的结合,使已发生特异性克隆扩增的T细胞发生自身凋亡而数量下降。因此,适度的T细胞活化可能作为HIV治疗的一种方法和策略。2007年上半年,Cell杂志的文章报道从人血液透析液中分离的小分子多肽(VIRIP)及其衍生物能够与HIV-1的FP特异性结合并抑制HIV包膜蛋白介导的病毒进入过程。而Shai Y在J Clin Invest曾发表论文,报道作为HIV-1的组成多肽,gp41 FP除了与病毒进入密切相关,还能直接影响免疫细胞活化。在上述的研究基础上,我们提出了尚未解答的问题:通过与FP结合而抑制HIV病毒进入的VIRIP衍生物,会不会对T细胞活化产生影响?其作用机制又是什么?
为此,本课题根据我们在HIV和SARS病毒进入抑制剂研究领域积累的经验,以AIV和HIV作为研究对象,为研究AIV病毒进入抑制剂和HIV杀微生物剂奠定研究基础。(1)靶向H5N1禽流感病毒血凝素亚基HA2的病毒进入抑制剂研究:建立抑制H5N1型AIV六螺旋束结构和N-端帽子结构形成的高通量筛选模型,并建立H5N1型禽流感假病毒模型,筛选小分子化合物库,再利用活病毒感染MDCK模型和活病毒感染小鼠模型,验证筛选出的小分子化合物的抗病毒活性,寻找能有效抑制H5N1型AIV进入靶细胞,且具有良好膜通透性的活性分子,为开发抗禽流感病毒进入抑制剂类药物奠定基础。(2)抗HIV多肽VIR576对抗原特异性T细胞活化的影响:采用T细胞活化的检测方法,研究VIR576对抗原特异性T细胞活化的影响和作用机制。由于HIV感染与T细胞关系密切,研究将有助于初步探讨VIR576与T细胞的关系,并分析其对HIV病毒感染的可能影响,有助于评价VIR576作为抗HIV杀微生物剂的可能性和机制。
第一部分靶向H5N1禽流感病毒血凝素亚基HA2的病毒进入抑制剂研究
目的:
建立抑制H5N1型AVI六螺旋束结构和N-端帽子结构形成的高通量筛选方法,并建立H5N1型禽流感假病毒模型,筛选小分子化合物库,得到小分子抗AIV化合物,再利用H5N1型活病毒感染MDCK模型和H5N1型活病毒感染小鼠模型进行验证,寻找能有效抑制H5N1型AIV感染的活性分子,研究其抗禽流感的作用。
方法:
1.合成衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽,包括N-多肽(N29,aa77～aa105)、C-多肽(C19,aa110～aa128)以及用FITC标记的荧光C-多肽、生物素标记的N-末端帽子多肽(N8,aa34～aa37)、FITC标记的C-末端帽子多肽(C8,aa173～aa176)。通过FLISA、荧光天然凝胶电泳、分子筛高效液相色谱等方法研究两组多肽间的相互作用,尝试建立阻止六螺旋束结构形成的荧光高通量筛选方法、抑制N-端帽子形成的高通量筛选方法、阻止六螺旋束结构形成的荧光天然凝胶电泳和分子筛高效液相色谱。
2.建立细胞水平的H5N1型禽流感假病毒活性检测方法:禽流感病毒(Avianinfluenza virus,AIV)的包膜蛋白有两个,分别为血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA),将表达这两个包膜蛋白的质粒与pNL43-Luc.R~-E~-(带有荧光素酶报告基因的HIV-1基因)共转染至293 T细胞后,细胞表达禽流感病毒包膜蛋白(HA、NA)和HIV-1颗粒,它们可被组装成为HA-NA/HIV假病毒,用来筛选作用于禽流感病毒包膜蛋白的化合物。
3.抗流感药物活性验证:应用H5N1型活病毒感染MDCK细胞模型和H5N1型活病毒感染Balb/c鼠模型进行活性验证,检测药物对病毒感染的MDCK细胞的保护作用及对MDCK细胞的毒性(CPE观察及XTT法检测)、药物对病毒感染动物的死亡保护作用等,评价抗流感病毒新药。即XTT法检测药物对MDCK细胞的毒性,CPE法(细胞病变)检测药物对流感病毒感染的MDCK细胞的保护作用,流感病毒对小鼠半数致死量(LD50)的测定和药物对流感病毒感染小鼠的死亡保护试验。
4.建立小鼠血浆中ARC-36的HPLC检测方法,研究ARC-36在小鼠体内的药代动力学,考察小鼠腹腔注射ARC-36的急性毒性。
结果:
1.FLISA、荧光天然凝胶电泳、分子筛高效液相色谱等方法均表明,衍生于H5N1高致病性禽流感病毒HA2的两组多肽(N29与C19、N8与C8)之间没有相互作用。
2.假病毒体系HA-NA/HIV可感染293T细胞和MDCK细胞,筛选出的化合物ARC-36抑制H5N1禽流感假病毒活性的IC50值为4.00±0.38μM,且作用在病毒进入阶段;合成的衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽,没有抑制H5N1禽流感的活性。
3.在MDCK细胞模型中,随着药物浓度的增加,病毒的毒性(血凝单位)呈下降趋势,其IC50为27.03μM。MDCK细胞毒性实验显示ARC-36的CC50为525.4μM。滴鼻感染前滴鼻给药组和感染后腹腔注射8天组小鼠的平均存活时间分别为8.8天和8.6天,与感染前正常对照组比较差异有统计学意义(p＜0.01)。
4.在小鼠血浆中ARC-36的HPLC测定法中(A相100%甲醇,B相0.5%冰醋酸梯度洗脱),ARC-36的保留时间为6.3 min,标准曲线方程为Y=5354.2X+7106.5,r=0.9993(n=5)。小鼠腹腔静脉注射ARC-36(50mg·kg~(-1))后血浆C-t曲线呈二室模型。从药代动力学参数可知,ARC-36消除半衰期(t_(1/2))_β为31.6 min。小鼠腹腔注射ARC-36的LD50为149.2 mg·kg~(-1)(7.5 mg·ml~(-1),211.8μM)。
结论:
1.人工合成的衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽没有抑制H5N1禽流感的活性。采用FLISA、圆二色谱、荧光天然凝胶电泳、分子筛高效液相色谱等技术发现,各组多肽之间没有相互作用,无法建立抑制H5N1型AVI六螺旋束结构和N-端帽子结构形成的高通量筛选方法。
2.建立的假病毒体系HA-NA/HIV可高效感染多种细胞系,筛选出的化合物ARC-36抑制H5N1禽流感假病毒活性较高,且作用于病毒进入阶段。
3.ARC-36对H5N1型禽流感病毒有较强的抑制作用,且ARC-36滴鼻或腹腔注射均可以延长小鼠的存活时间,但对小鼠存活率没有明显的保护作用,可能原因是ARC-36的半衰期较短。
4.本实验建立的小鼠血浆中ARC-36的HPLC测定法操作简便、快速、准确,可用于ARC-36的体内定量分析。小鼠腹腔静脉注射ARC-36后血浆C-t曲线呈二室模型,ARC-36在血中清除迅速。小鼠腹腔注射ARC-36具有一定的安全性。
第二部分抗HIV多肽VIR576对抗原特异性T细胞活化的影响及机制研究
目的:
采用T细胞活化的检测方法,研究VIR576对抗原特异性T细胞活化的作用,探讨其对T细胞活化及HIV免疫的影响;并用生物物理等方法研究VIR576与TCR-TMD之间的相互作用,评价VIR576作为HIV杀微生物剂的可能性和机制。
方法:
1.MOG35-55体外刺激抗原特异性T细胞系A2b细胞,[~3H]脱氧胸苷摄入法测定VIR576对A2b细胞活化的影响;OVA体外刺激分离的DO11.10小鼠抗原特异性T细胞,CCK-8法测定VIR576对其活化的影响;刀豆蛋白(ConA)或抗鼠CD3抗体体外刺激非抗原特异性小鼠T细胞,CCK-8法测定VIR576对其活化的影响;采用免疫磁珠两步法分离小鼠脾组织CD4~+CD25~- T细胞,CCK-8法测定VIR576对其活化的影响。
2.用溶血试验、溶血抑制实验、FRET、FLISA、竞争性FLISA等方法检测VIR576与TCR-TMD之间的相互作用,并研究其主要的结合区域。多肽TCR-TMD加入到人红细胞悬液,在405 nm处测吸光度,考察多肽TCR-TMD的插膜作用;多肽VIR576或VIR-scram与多肽TCR-TMD孵育后加入到人红细胞悬液,考察其对多肽TCR-TMD插膜作用的抑制作用。TCR-TMD包被到96孔酶标板里,加入相应浓度的Rho-VIR576,;或者加入Rho-VIR576和未标记的VIR576,读取各孔荧光密度值,考察TCR-TMD与VIR576之间的相互作用。用FRET技术考察TCR-TMD的核心序列CP与VIR576的相互关系,将CP-NBD加入到LUV溶液,在467 nm波长激发,500 nm-600 nm波长发射,再加入Rho-VIR576,考察其能量转移(FRET)。
3.BALB/c小鼠脾细胞用抗鼠-CD3抗体刺激72小时后,4%多聚甲醛冰上固定,加入1:50稀释的兔抗鼠CD4抗体,再加入1:50稀释的标记的羊抗兔IgG,在孵育的最后5 min加入Rho-VIR576。细胞用PBS洗三次,在激光共聚焦显微镜下观察VIR576在T细胞细胞膜的定位和与TCR分子的共分布情况。
结果:
1.体外实验显示,用MOG35-55(10μM)可以刺激出明显的T细胞活化,VIR576(50μM)能抑制MOG35-55抗原特异性T细胞活化,并且能逆转FP(50μM)对抗原特异性T细胞活化的抑制作用(p＜0.05);对OVA抗原特异性T淋巴细胞,VIR576(50μM)同样能抑制抗原特异性T细胞活化(p＜0.05),并且能反转FP(50μM)介导的抗原特异性T细胞活化;但VIR576对非抗原特异性T细胞活化没有作用;VIR576同样对OVA刺激的抗原特异性CD4~+CD25~-T淋巴细胞增殖表现出显著的抑制作用。
2.VIR576与TCR-TMD的核心区域CP(aa253-261)结合:VIR576较低浓度即能抑制TCR-TMD介导的溶血,其IC50为1.23±0.16μM,但是VIRIP的乱序(VIR-scram)不能抑制TCR-TMD介导的溶血(p＜0.05);FLISA实验结果显示,Rho-VIR576能与TCR-TMD(2μM)结合,且随着浓度增加(2μM-64μM),Rho-VIR576结合的越多,荧光强度越高;竞争性FLISA实验结果显示,未标记的VIR576加入越多,Rho-VIR576结合的越少,荧光强度越低。FRET实验也显示NBD-CP与Rho-VIR576之间存在能量转移,且呈剂量关系。
3.荧光共聚焦显微镜观察到VIR576不能与未活化的CD4~+T细胞结合,但能结合到活化的CD4~+T细胞膜表面的CD4分子,且CD4~+T细胞活化的区域才能标记上荧光。
结论:
1.VIR576能抑制抗原特异性T细胞活化,也能反转FP介导的抗原特异性T细胞活化,但对非抗原特异性T细胞活化没有作用。
2.溶血实验显示VIR576能抑制TCR-TMD介导的红细胞溶血,FLISA及竞争性FLISA法结果提示VIR576能与TCR-TMD特异性的结合,FRET实验证实VIR576能与TCR-TMD的主要区域CP结合。
3.VIR576能结合到活化的T细胞膜表面,且与CD4分子结合,间接证明其能与T细胞表面的TCR结合。
Background:
Acquiredimmunodeficiency syndrome(AIDS) and influenza are the major threats to public health worldwide.Similar to the SARS coronavirus(SARS-CoV), human immunodeficiency virus(HIV) and avian influenza virus(AIV) are also classⅠenveloped virus.ClassⅠenveloped viruses share a similar virus-host cell membrane fusion mechanism:the envelope glycoprotein(Env) binds to the host cell receptor and undergoes conformational changes which trigger the membrane fusion.Using HIV and influenza virus as examples,the membrane fusion is mediated by the Envs of influenza virus(hemagglutinin,HA0) and of HIV(gp160).Both envelope proteins consist of two associated parts:the surface subunit(HA1/gp120) responsible for binding receptor and the transmembrane subunit(HA2/gp41) medaiteing membrane fusion.
A peptide HIV entry inhibitor derived from the HIV-1 gp41 can bind to its conterpart in gp41,thereby preventing further conformation change of gp41 and interfering with the virus fusion,which provide new ideas and strategies to the treatment of viral diseases.However,the peptide derived from the HA2 of AIV exhibits no inhibitory activity against influenza virus.This is because AIV has a fusogenic mechanism different from that mediated by the HIV gp41.Fusion of the AIV envelope with the host cell membranes takes place in endosomal compartments, whilethese initial stages of virus replication are dependent on the acidification of endosomes by cellular proton pumps.The reduction in endosomal pH triggers the conformational changes in HA that lead to membrane fusion.Most of the active antiviral peptides derived from the virus envelope proteins contain more 30 amino acid residues.However,the peptide with large molecular size peptides is difficult to enter through the cell membrane to the intracellular compartments to interact with the influenza virus HA2.Even a peptide is able to enter the endosome,it may be inactivated by low pH(about 5) in the acidic environment.In contrast,many small molecular compounds can enter into the cell through the cellular membrane. Therefore,a small molecule compound that can prevent the influenza virus HA2 conformational change may be effective ininhibiting influenza virus infection.
HIV,a lentivirus,is the causative agent of AIDS,a disease that progressively reduces the effectiveness of human immune system,leading to life-threatening opportunistic infections and tumors.HIV primarily infects vital cells in the human immune system such as CD4~+ T helper(Th) cells.To develop effective therapeutics and vaccines for treatement and prevention of AIDS,it is critical to understand pathogenesis of HIV infection and the interaction between virus and immune cells, especially T-cell activation mediated by viral proteins.
T-cell activation is a double-edged sword in the pathogenesis of HIV/AIDS. On the one hand,activation of CD4~+ T cells is required to eliminate the infected viruses because the activated CD4~+ Th cells can mediate humoral and cellular immune responses against HIV-1.On the other hand,HIV-1 preferentially infects the activated CD4~+ T cells,especially those in mucosa.Abnormal CD4~+ T-cell activation caused by HIV-1 infection contributes to activation-induced cell apoptosis and precedes the host immune system crash.Accordingly,proper regulation of CD4~+ T-cell activation may be applied as a therapeutic or prophylactic strategy for treatment or prevention of HIV-1 infection.Munch et al have reported that a 20-mer peptide derived from the C-proximal subfragment of al-antitrypsin(al-AT), designated Virus Inhibitory Peptide(VIRIP),is able to interact with the HIV-1 gp41 FP and inhibit HIV-1 Env-mediated cell-cell fusion.Its analog peptide,VIR576,is about two orders of magnitude more potent than VIRIP in inhibiting HIV-1 replication.Given that FP could effectively downregulate the T-cell activation as proven by Shai and colleagues,I thus hypothesized that the VIRIP may also affect T cell activation and may be applied as a dual functional anti-HIV agent.
In the present study,I focused on two projects:(1) Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutinin HA2 subunit;and(2) Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptor.For the first project,I intended to establish:(ⅰ) an FLISA-based primary HTS assay for screening compounds that can block six-helix bundle formation between the NHR and CHR domains and that can inhibit the formation of the N-terminal cap structure in HA2 of AIV;(ⅱ) a cell-based secondary HTS assay for screening compounds that inhibit infection of MDCK cells by H5N1 pseudotyped viruses;and(ⅲ) a mouse model for evaluating the in vivo efficacy and toxicity of the potential anit-AIV compounds.For the second project,I intended to study:(ⅰ) the effect of VIR576 on the proliferation of T cells in response to specific and non-specific antigen stimuation;(ⅱ) the effect of VIR576 on TCR-TMD-mediated hemolysis;and(ⅲ) whether VIR576 is able to colocalize with CD4 molecule in the CD4~+ T cell membrane to interact with TCR.
PartⅠ.Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutiniu HA2 subunit
OBJECTIVE:
To establish the high-throughput screening methods for screening the compounds inhibited formation of the six-helix bundle and the N-terminal cap structure in the HA2 domain of AIV,and to develop a cell-based pharmacological model by using the H5N1 pseudotyped viral system,a MDCK cell infected model for in vitro infection by live H5N1 virus,and a mouse model for in vivo infection by live H5N1 virus,and to screen compounds which can specifically inhibit the virus entry.
METHODS:
1.To synthesize a series of peptides derived from the HA2 domain of the highly pathogenic H5N1 avian influenza virus,including an N-peptide(N29, aa77～aa105),a C-peptide(C19,aa110～aa128) and an FITC-labeled C-peptide,a biotin-labeled N-terminal cap peptide(N8,aa34～aa37),and an FITC-labeled C-terminal cap peptide(C8,aa173～aa176).The potential interactions between the N- and C-peptides and between N- and C-terimal cap peptides were studies by using FLISA,natural fluorescence gel electrophoresis,SE-HPLC methods,in order to establish the high-throughput screening methods for screening of compounds that can inhibit formation of the six-helix bundle formation and the N-terminal cap structure in HA2 of AIV.
2.To establish a cell-based pharmacological model for compounds screening by using the H5N1 pseudotyped viral system.The plasmids encoding the AIV Envs HA and NA and the pNL43Luc.RE-(with a luciferase reporter gene and a HIV-1 core gene) were transfected into 293T cells for expression of HA and NA on pseudotyped virus containing the HIV-1 core.This system would be used for screening of anti-AIV compounds.
3.To develop in vitro H5N1 AIV infection model using MDCK cells and in vivo H5N1 AIV infection model using the Balb/c mice for evaluation of the in vitro and in vivo efficacy of anti-AIV compounds.
4.To establish an XTT assay and a mouse model for evaluating the in vitro and in vivo toxicity,respectively,of the selected anti-AIV compounds.
5.To establish an HPLC method for studying the pharmacokinetics of ARC-36 in mice.
RESULTS:
1.The results from FLISA,natural fluorescence gel electrophoresis,SE-HPLC analysis showed that there were no detectable interactions between N29 and C19 and between N8 and C8 peptides derived from the HA2 domain of H5N1 AIV.
2.The HA-NA/HIV pseudovirus could infect 293T cells and MDCK cells,and ARC-36 could inhibit the AIV pseudovirus infection with IC_(50) value of 4.00±0.38μM.
3.ARC-36 also inhibited live H5N1 virus infection of MDCK cells with IC50 of 27.03±2.54μM.It had now toxicity to MDCK cells with CC_(50) of 525.4±63.22μM.Intranasal administration of ARC-36 before the intranasal infection protected mice from H5N1 AIV challenge,with the average survival time of 8.8 days,while 7.4 days for the control normal mice,a significant differences(p＜0.01).
4.As analyszed by HPLC(A phase:100%methanol,B phase:0.5%glacial acetic acid),the retention time of ARC-36 was 6.3 min with a standard curve equation of Y= 5354.2X+7106.5,r = 0.9993(n = 5).The Ct curve is two-compartment model after intraperitoneal injection of ARC-36.The elimination half-life(t1/2)_βis 31.6min.The LD50 of ARC-36 by intraperitoneal injection is 149.2 mg·kg~(-1) (7.5mg·ml~(-1),211.8μM),and is about 50-fold of the IC_(50) value of ARC-36 determed in pseudovirus assay(4.00±0.38μM).
CONCLUSION:
1.Because of no detectable interactions between N29 and C19 and between N8 and C8,we failed to develop these peptides-based high-throughput screening methods for screening of the compounds that inhibit formation of six-helix bundle and the N-terminal cap structure in HA2 of H5N1 AIV.
2.I successfully developed the HA-NA/HIV pseudovirus infection system using 293T cells and MDCK cells for screening of anti-AIV compounds and using this system,we found that ARC-36 was effective in inhibiting AIV infection,but was infective when it was added 24 hours post infection.
3.I established the Balb/c mouse AIV challenge model and MDCK cell H5N1 AIV infection model for evaluating the in vivo and in vitro efficacy of the selected anti-AIV compounds.We confirmed that ARC-36 is effective in inhibiting H5N1 AIVinfection in MDCK cells.Intranasal administration of ARC-36 before the intranasal infection resulted in prolonged average survival time of mice.
4.I established the HPLC method for quantitative analysis of ARC-36 level in vivo. I found that he Ct curve is two-compartment model after intraperitoneal injection of ARC-36,and the elimination half-life(t1/2)_βis 31.6 min,owing to the quick metabolism.The LD50 of ARC-36 by intraperitoneal injection is 50-fold of the IC50 value determined in pseudovirus assay,suggesting that ARC-36 is relatively safe.
PARTⅡ.Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptor
OBJECTIVE:
To study the effect of VIR576 on antigen-specific T cell(Ab2 cells,CD4~+ CD25T cells) activation and non-antigen-specific T cell activation;To analyze the membrane distribution of VIR576 in T cell using the fluorescent confocal microscope and the colocalization of VIR576 with TCR;To study the interaction of VIR576 and the synthetic peptide derived from TCR-TMD,the CP sequence(aa 253-261),using hemolysis test,hemolysis inhibition assay,FRET,FLIA,and competitive FLISA.
METHODS:
1.To investigate the potential effect of FP16,VIR576,and the mixture of FP16 and VIR576 on the proliferation of A2b cells in response to MOG 35-55 at 10μg/ml, to test the effect of FP16,VIR576 and their mixture on proliferation of splenocytes of OVA-specific DO 11.10 Tg mice in response to chicken OVA,and to test the proliferation activity of non-stimulated and Con A-stimulated splenocytes from BALB/c.
2.To establish TCR-TMD-mediated hemolysis assay,and using this assay,to study the effect of VIR576 on the hemolytic activity of TCR-TMD.
3.To develop a simple and rapid fluorescence-based binding assay using Rhodamine(Rho)-conjugated VIR576 to evaluate the potential interaction between VIR576 and TCR-TMD,to conduct a competitive assay to confirm the specific interaction between TCR-TMD and Rho-VIR576 using unlabeled-VIR576 and VIRIP scramble peptide,and to identify the critical binding site of VIR576 in TCR-TMD.
4.4.To study whether VIR576 could colocalize with CD4 molecule in the CD4~+ T cell membrane to interact with TCR.
RESULTS:
1.VIR576 alone suppressed the proliferation of A2b T cells in responses to MOG 35-55,and it could also block FP16-mediated inhibition of A2b cell activation (p＜0.05).VIR576 itself also inhibited splenocytes proliferation in responses to OVA,and it could block FP16-mediated inhibition of splenocytes activation (p＜0.05).Both splenocytes and CD4~+CD25~-T cells of DO11.10 mice showed a dose-dependent strong proliferative in response to the stimulation by OVA,while the addition of VIR576 resulted in significant inhibition of proliferation of splenocytes and the primary CD4~+CD25~- T cells of DO11.10 mice.The proliferation of non-stimulated and Con A-stimulated splenocytes was not affected by addition of VIR576 at the concentration as high as 50μg/ml. Consistently,VIR576 had no significant effect on proliferation of DO11.10 mouse splenocytes in the absence or presence of Con A or anti-CD3 antibody.
2.VIR576 interacted with CP since incubation of human erythrocytes with TCR-TMD resulted in significant hemolysis in a dose-dependant manner with IC_(50) of 1.23±0.16μM,whereas the VIRIP scramble peptide had no significant inhibition on TCR-TMD-mediated hemolysis(p＜0.05).Rho-VIR576 bound to TCR-TMD in a dose-dependant manner,resulting in a significant increase of the fluorescence intensity.A significant decrease in fluorescence intensity when mixture of unlabeled-VIR576 and Rho-VIR576 were added to the immobilized TCR-TMD,while the unlabeled VIRIP scramble peptide had no effect on the binding of Rho-VIR576 to TCR-TMD(p＜0.05).When Rho-VIR576(final concentration 0.04-0.12μM) was added to a mixture of NBD-CP(0.04μM) and PC LUV liposome(100μM),a dose-dependent quenching of the donor's emission,which is consistent with energy transfer,was observed.
3.Rho-VIR576 did not bind to the unstimulated lymphocytes,but strongly interacted with the CD4~+ T cells stimulated with anti-CD3 antibody.Only a part of activated T cell membrane was stained by Alexa-labeled anti-CD4 antibody.
CONCLUSION:
1.VIR576 was effective in suppressing the antigen-specific T cell activation,but has no effect on non-specific T cell proliferation.
2.VIR576 had the ability to downregulate the antigen-specific T cell activation by interaction with TCR transmembrane domain.
3.Rho-VIR576 strongly interacted with the CD4~+ T cells stimulated with anti-CD3 antibody.
艾滋病和禽流感成为当今中国乃至全世界有深远影响的重大流行疾病,是当前人类健康面临的重大威胁,但一直以来缺乏有效的治疗药物和疫苗。与SARS冠状病毒(SARS-CoV)相似,人类免疫缺陷病毒(HIV)和禽流感病毒(AIV)均为Ⅰ型包膜病毒,采用相似的病毒-宿主细胞膜融合机制,即病毒表面糖蛋白结合到宿主细胞受体后,启动病毒融合蛋白的一系列构象变化。包膜病毒进入靶细胞的过程需要病毒膜糖蛋白的介导,这一类型病毒的包膜蛋白一般都具有两个亚基,其中不跨膜的亚基(如HIV的gp120、AIV的HA1等)具有与靶细胞膜上病毒受体相结合的位点,而跨膜亚基(如HIV的gp41、AIV的HA2等)则介导病毒膜与靶细胞膜的融合。以上述研究为基础设计的病毒进入抑制剂,可以在病毒包膜糖蛋白中间体构象形成的短时间内,高效、特异地竞争结合其配体,从而阻止包膜糖蛋白的进一步折叠,达到抑制病毒入侵的目的,为病毒疾病的防治提供了新思路和策略。
虽然流感病毒也是Ⅰ型包膜病毒,但衍生自HA2的多肽不具有抑制AIV的活性,其原因可能与流感病毒本身的融合机制有关。流感病毒与靶细胞的融合发生在胞内体膜上,需要酸性环境的诱导。而衍生于包膜病毒的多肽,一般都要30个左右氨基酸残基的多肽才有效。多肽分子量大,很难通过细胞膜进入到胞内体与流感病毒HA2的相应区域结合,另外,即使进到胞内体中去了,pH为5左右的酸性环境对多肽的活性可能也具有破坏作用。相比之下,许多小分子化合物能够轻易地穿过靶细胞膜并进入胞内体,若小分子化合物能阻止流感病毒HA2构象的改变,就有可能具有抗流感病毒感染的作用。
HIV是一种感染人类免疫系统细胞的慢病毒,主要感染CD4~+T淋巴细胞,特征性的引起CD4~+T细胞逐步减少和进行性免疫缺陷,最后导致艾滋病(获得性免疫缺陷综合征),引起机会性感染并最终导致死亡。病毒与宿主免疫细胞的相互作用是影响疾病发展的重要因素。在HIV感染导致的AIDS疾病进展过程中,T细胞活化是一把双刃剑。一方面CD4~+T细胞的活化在清除病毒过程是非常必要的,成熟的CD4~+Th细胞是抗病毒免疫的关键因素。另一方面,HIV膜蛋白(Env)能结合CD4~+受体,在CD4~+T细胞中复制,由于病毒颗粒的持续表达,HIV感染导致T细胞增殖增加,T细胞活化后又可借助自身表达的凋亡受体与配体的结合,使已发生特异性克隆扩增的T细胞发生自身凋亡而数量下降。因此,适度的T细胞活化可能作为HIV治疗的一种方法和策略。2007年上半年,Cell杂志的文章报道从人血液透析液中分离的小分子多肽(VIRIP)及其衍生物能够与HIV-1的FP特异性结合并抑制HIV包膜蛋白介导的病毒进入过程。而Shai Y在J Clin Invest曾发表论文,报道作为HIV-1的组成多肽,gp41 FP除了与病毒进入密切相关,还能直接影响免疫细胞活化。在上述的研究基础上,我们提出了尚未解答的问题:通过与FP结合而抑制HIV病毒进入的VIRIP衍生物,会不会对T细胞活化产生影响?其作用机制又是什么?
为此,本课题根据我们在HIV和SARS病毒进入抑制剂研究领域积累的经验,以AIV和HIV作为研究对象,为研究AIV病毒进入抑制剂和HIV杀微生物剂奠定研究基础。(1)靶向H5N1禽流感病毒血凝素亚基HA2的病毒进入抑制剂研究:建立抑制H5N1型AIV六螺旋束结构和N-端帽子结构形成的高通量筛选模型,并建立H5N1型禽流感假病毒模型,筛选小分子化合物库,再利用活病毒感染MDCK模型和活病毒感染小鼠模型,验证筛选出的小分子化合物的抗病毒活性,寻找能有效抑制H5N1型AIV进入靶细胞,且具有良好膜通透性的活性分子,为开发抗禽流感病毒进入抑制剂类药物奠定基础。(2)抗HIV多肽VIR576对抗原特异性T细胞活化的影响:采用T细胞活化的检测方法,研究VIR576对抗原特异性T细胞活化的影响和作用机制。由于HIV感染与T细胞关系密切,研究将有助于初步探讨VIR576与T细胞的关系,并分析其对HIV病毒感染的可能影响,有助于评价VIR576作为抗HIV杀微生物剂的可能性和机制。
第一部分靶向H5N1禽流感病毒血凝素亚基HA2的病毒进入抑制剂研究
目的:
建立抑制H5N1型AVI六螺旋束结构和N-端帽子结构形成的高通量筛选方法,并建立H5N1型禽流感假病毒模型,筛选小分子化合物库,得到小分子抗AIV化合物,再利用H5N1型活病毒感染MDCK模型和H5N1型活病毒感染小鼠模型进行验证,寻找能有效抑制H5N1型AIV感染的活性分子,研究其抗禽流感的作用。
方法:
1.合成衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽,包括N-多肽(N29,aa77～aa105)、C-多肽(C19,aa110～aa128)以及用FITC标记的荧光C-多肽、生物素标记的N-末端帽子多肽(N8,aa34～aa37)、FITC标记的C-末端帽子多肽(C8,aa173～aa176)。通过FLISA、荧光天然凝胶电泳、分子筛高效液相色谱等方法研究两组多肽间的相互作用,尝试建立阻止六螺旋束结构形成的荧光高通量筛选方法、抑制N-端帽子形成的高通量筛选方法、阻止六螺旋束结构形成的荧光天然凝胶电泳和分子筛高效液相色谱。
2.建立细胞水平的H5N1型禽流感假病毒活性检测方法:禽流感病毒(Avianinfluenza virus,AIV)的包膜蛋白有两个,分别为血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA),将表达这两个包膜蛋白的质粒与pNL43-Luc.R~-E~-(带有荧光素酶报告基因的HIV-1基因)共转染至293 T细胞后,细胞表达禽流感病毒包膜蛋白(HA、NA)和HIV-1颗粒,它们可被组装成为HA-NA/HIV假病毒,用来筛选作用于禽流感病毒包膜蛋白的化合物。
3.抗流感药物活性验证:应用H5N1型活病毒感染MDCK细胞模型和H5N1型活病毒感染Balb/c鼠模型进行活性验证,检测药物对病毒感染的MDCK细胞的保护作用及对MDCK细胞的毒性(CPE观察及XTT法检测)、药物对病毒感染动物的死亡保护作用等,评价抗流感病毒新药。即XTT法检测药物对MDCK细胞的毒性,CPE法(细胞病变)检测药物对流感病毒感染的MDCK细胞的保护作用,流感病毒对小鼠半数致死量(LD50)的测定和药物对流感病毒感染小鼠的死亡保护试验。
4.建立小鼠血浆中ARC-36的HPLC检测方法,研究ARC-36在小鼠体内的药代动力学,考察小鼠腹腔注射ARC-36的急性毒性。
结果:
1.FLISA、荧光天然凝胶电泳、分子筛高效液相色谱等方法均表明,衍生于H5N1高致病性禽流感病毒HA2的两组多肽(N29与C19、N8与C8)之间没有相互作用。
2.假病毒体系HA-NA/HIV可感染293T细胞和MDCK细胞,筛选出的化合物ARC-36抑制H5N1禽流感假病毒活性的IC50值为4.00±0.38μM,且作用在病毒进入阶段;合成的衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽,没有抑制H5N1禽流感的活性。
3.在MDCK细胞模型中,随着药物浓度的增加,病毒的毒性(血凝单位)呈下降趋势,其IC50为27.03μM。MDCK细胞毒性实验显示ARC-36的CC50为525.4μM。滴鼻感染前滴鼻给药组和感染后腹腔注射8天组小鼠的平均存活时间分别为8.8天和8.6天,与感染前正常对照组比较差异有统计学意义(p＜0.01)。
4.在小鼠血浆中ARC-36的HPLC测定法中(A相100%甲醇,B相0.5%冰醋酸梯度洗脱),ARC-36的保留时间为6.3 min,标准曲线方程为Y=5354.2X+7106.5,r=0.9993(n=5)。小鼠腹腔静脉注射ARC-36(50mg·kg~(-1))后血浆C-t曲线呈二室模型。从药代动力学参数可知,ARC-36消除半衰期(t_(1/2))_β为31.6 min。小鼠腹腔注射ARC-36的LD50为149.2 mg·kg~(-1)(7.5 mg·ml~(-1),211.8μM)。
结论:
1.人工合成的衍生于H5N1高致病性禽流感病毒血凝素亚基HA2的多肽没有抑制H5N1禽流感的活性。采用FLISA、圆二色谱、荧光天然凝胶电泳、分子筛高效液相色谱等技术发现,各组多肽之间没有相互作用,无法建立抑制H5N1型AVI六螺旋束结构和N-端帽子结构形成的高通量筛选方法。
2.建立的假病毒体系HA-NA/HIV可高效感染多种细胞系,筛选出的化合物ARC-36抑制H5N1禽流感假病毒活性较高,且作用于病毒进入阶段。
3.ARC-36对H5N1型禽流感病毒有较强的抑制作用,且ARC-36滴鼻或腹腔注射均可以延长小鼠的存活时间,但对小鼠存活率没有明显的保护作用,可能原因是ARC-36的半衰期较短。
4.本实验建立的小鼠血浆中ARC-36的HPLC测定法操作简便、快速、准确,可用于ARC-36的体内定量分析。小鼠腹腔静脉注射ARC-36后血浆C-t曲线呈二室模型,ARC-36在血中清除迅速。小鼠腹腔注射ARC-36具有一定的安全性。
第二部分抗HIV多肽VIR576对抗原特异性T细胞活化的影响及机制研究
目的:
采用T细胞活化的检测方法,研究VIR576对抗原特异性T细胞活化的作用,探讨其对T细胞活化及HIV免疫的影响;并用生物物理等方法研究VIR576与TCR-TMD之间的相互作用,评价VIR576作为HIV杀微生物剂的可能性和机制。
方法:
1.MOG35-55体外刺激抗原特异性T细胞系A2b细胞,[~3H]脱氧胸苷摄入法测定VIR576对A2b细胞活化的影响;OVA体外刺激分离的DO11.10小鼠抗原特异性T细胞,CCK-8法测定VIR576对其活化的影响;刀豆蛋白(ConA)或抗鼠CD3抗体体外刺激非抗原特异性小鼠T细胞,CCK-8法测定VIR576对其活化的影响;采用免疫磁珠两步法分离小鼠脾组织CD4~+CD25~- T细胞,CCK-8法测定VIR576对其活化的影响。
2.用溶血试验、溶血抑制实验、FRET、FLISA、竞争性FLISA等方法检测VIR576与TCR-TMD之间的相互作用,并研究其主要的结合区域。多肽TCR-TMD加入到人红细胞悬液,在405 nm处测吸光度,考察多肽TCR-TMD的插膜作用;多肽VIR576或VIR-scram与多肽TCR-TMD孵育后加入到人红细胞悬液,考察其对多肽TCR-TMD插膜作用的抑制作用。TCR-TMD包被到96孔酶标板里,加入相应浓度的Rho-VIR576,;或者加入Rho-VIR576和未标记的VIR576,读取各孔荧光密度值,考察TCR-TMD与VIR576之间的相互作用。用FRET技术考察TCR-TMD的核心序列CP与VIR576的相互关系,将CP-NBD加入到LUV溶液,在467 nm波长激发,500 nm-600 nm波长发射,再加入Rho-VIR576,考察其能量转移(FRET)。
3.BALB/c小鼠脾细胞用抗鼠-CD3抗体刺激72小时后,4%多聚甲醛冰上固定,加入1:50稀释的兔抗鼠CD4抗体,再加入1:50稀释的标记的羊抗兔IgG,在孵育的最后5 min加入Rho-VIR576。细胞用PBS洗三次,在激光共聚焦显微镜下观察VIR576在T细胞细胞膜的定位和与TCR分子的共分布情况。
结果:
1.体外实验显示,用MOG35-55(10μM)可以刺激出明显的T细胞活化,VIR576(50μM)能抑制MOG35-55抗原特异性T细胞活化,并且能逆转FP(50μM)对抗原特异性T细胞活化的抑制作用(p＜0.05);对OVA抗原特异性T淋巴细胞,VIR576(50μM)同样能抑制抗原特异性T细胞活化(p＜0.05),并且能反转FP(50μM)介导的抗原特异性T细胞活化;但VIR576对非抗原特异性T细胞活化没有作用;VIR576同样对OVA刺激的抗原特异性CD4~+CD25~-T淋巴细胞增殖表现出显著的抑制作用。
2.VIR576与TCR-TMD的核心区域CP(aa253-261)结合:VIR576较低浓度即能抑制TCR-TMD介导的溶血,其IC50为1.23±0.16μM,但是VIRIP的乱序(VIR-scram)不能抑制TCR-TMD介导的溶血(p＜0.05);FLISA实验结果显示,Rho-VIR576能与TCR-TMD(2μM)结合,且随着浓度增加(2μM-64μM),Rho-VIR576结合的越多,荧光强度越高;竞争性FLISA实验结果显示,未标记的VIR576加入越多,Rho-VIR576结合的越少,荧光强度越低。FRET实验也显示NBD-CP与Rho-VIR576之间存在能量转移,且呈剂量关系。
3.荧光共聚焦显微镜观察到VIR576不能与未活化的CD4~+T细胞结合,但能结合到活化的CD4~+T细胞膜表面的CD4分子,且CD4~+T细胞活化的区域才能标记上荧光。
结论:
1.VIR576能抑制抗原特异性T细胞活化,也能反转FP介导的抗原特异性T细胞活化,但对非抗原特异性T细胞活化没有作用。
2.溶血实验显示VIR576能抑制TCR-TMD介导的红细胞溶血,FLISA及竞争性FLISA法结果提示VIR576能与TCR-TMD特异性的结合,FRET实验证实VIR576能与TCR-TMD的主要区域CP结合。
3.VIR576能结合到活化的T细胞膜表面,且与CD4分子结合,间接证明其能与T细胞表面的TCR结合。
Background:
Acquiredimmunodeficiency syndrome(AIDS) and influenza are the major threats to public health worldwide.Similar to the SARS coronavirus(SARS-CoV), human immunodeficiency virus(HIV) and avian influenza virus(AIV) are also classⅠenveloped virus.ClassⅠenveloped viruses share a similar virus-host cell membrane fusion mechanism:the envelope glycoprotein(Env) binds to the host cell receptor and undergoes conformational changes which trigger the membrane fusion.Using HIV and influenza virus as examples,the membrane fusion is mediated by the Envs of influenza virus(hemagglutinin,HA0) and of HIV(gp160).Both envelope proteins consist of two associated parts:the surface subunit(HA1/gp120) responsible for binding receptor and the transmembrane subunit(HA2/gp41) medaiteing membrane fusion.
A peptide HIV entry inhibitor derived from the HIV-1 gp41 can bind to its conterpart in gp41,thereby preventing further conformation change of gp41 and interfering with the virus fusion,which provide new ideas and strategies to the treatment of viral diseases.However,the peptide derived from the HA2 of AIV exhibits no inhibitory activity against influenza virus.This is because AIV has a fusogenic mechanism different from that mediated by the HIV gp41.Fusion of the AIV envelope with the host cell membranes takes place in endosomal compartments, whilethese initial stages of virus replication are dependent on the acidification of endosomes by cellular proton pumps.The reduction in endosomal pH triggers the conformational changes in HA that lead to membrane fusion.Most of the active antiviral peptides derived from the virus envelope proteins contain more 30 amino acid residues.However,the peptide with large molecular size peptides is difficult to enter through the cell membrane to the intracellular compartments to interact with the influenza virus HA2.Even a peptide is able to enter the endosome,it may be inactivated by low pH(about 5) in the acidic environment.In contrast,many small molecular compounds can enter into the cell through the cellular membrane. Therefore,a small molecule compound that can prevent the influenza virus HA2 conformational change may be effective ininhibiting influenza virus infection.
HIV,a lentivirus,is the causative agent of AIDS,a disease that progressively reduces the effectiveness of human immune system,leading to life-threatening opportunistic infections and tumors.HIV primarily infects vital cells in the human immune system such as CD4~+ T helper(Th) cells.To develop effective therapeutics and vaccines for treatement and prevention of AIDS,it is critical to understand pathogenesis of HIV infection and the interaction between virus and immune cells, especially T-cell activation mediated by viral proteins.
T-cell activation is a double-edged sword in the pathogenesis of HIV/AIDS. On the one hand,activation of CD4~+ T cells is required to eliminate the infected viruses because the activated CD4~+ Th cells can mediate humoral and cellular immune responses against HIV-1.On the other hand,HIV-1 preferentially infects the activated CD4~+ T cells,especially those in mucosa.Abnormal CD4~+ T-cell activation caused by HIV-1 infection contributes to activation-induced cell apoptosis and precedes the host immune system crash.Accordingly,proper regulation of CD4~+ T-cell activation may be applied as a therapeutic or prophylactic strategy for treatment or prevention of HIV-1 infection.Munch et al have reported that a 20-mer peptide derived from the C-proximal subfragment of al-antitrypsin(al-AT), designated Virus Inhibitory Peptide(VIRIP),is able to interact with the HIV-1 gp41 FP and inhibit HIV-1 Env-mediated cell-cell fusion.Its analog peptide,VIR576,is about two orders of magnitude more potent than VIRIP in inhibiting HIV-1 replication.Given that FP could effectively downregulate the T-cell activation as proven by Shai and colleagues,I thus hypothesized that the VIRIP may also affect T cell activation and may be applied as a dual functional anti-HIV agent.
In the present study,I focused on two projects:(1) Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutinin HA2 subunit;and(2) Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptor.For the first project,I intended to establish:(ⅰ) an FLISA-based primary HTS assay for screening compounds that can block six-helix bundle formation between the NHR and CHR domains and that can inhibit the formation of the N-terminal cap structure in HA2 of AIV;(ⅱ) a cell-based secondary HTS assay for screening compounds that inhibit infection of MDCK cells by H5N1 pseudotyped viruses;and(ⅲ) a mouse model for evaluating the in vivo efficacy and toxicity of the potential anit-AIV compounds.For the second project,I intended to study:(ⅰ) the effect of VIR576 on the proliferation of T cells in response to specific and non-specific antigen stimuation;(ⅱ) the effect of VIR576 on TCR-TMD-mediated hemolysis;and(ⅲ) whether VIR576 is able to colocalize with CD4 molecule in the CD4~+ T cell membrane to interact with TCR.
PartⅠ.Identification of influenza virus entry inhibitors targeting H5N1 Env hemagglutiniu HA2 subunit
OBJECTIVE:
To establish the high-throughput screening methods for screening the compounds inhibited formation of the six-helix bundle and the N-terminal cap structure in the HA2 domain of AIV,and to develop a cell-based pharmacological model by using the H5N1 pseudotyped viral system,a MDCK cell infected model for in vitro infection by live H5N1 virus,and a mouse model for in vivo infection by live H5N1 virus,and to screen compounds which can specifically inhibit the virus entry.
METHODS:
1.To synthesize a series of peptides derived from the HA2 domain of the highly pathogenic H5N1 avian influenza virus,including an N-peptide(N29, aa77～aa105),a C-peptide(C19,aa110～aa128) and an FITC-labeled C-peptide,a biotin-labeled N-terminal cap peptide(N8,aa34～aa37),and an FITC-labeled C-terminal cap peptide(C8,aa173～aa176).The potential interactions between the N- and C-peptides and between N- and C-terimal cap peptides were studies by using FLISA,natural fluorescence gel electrophoresis,SE-HPLC methods,in order to establish the high-throughput screening methods for screening of compounds that can inhibit formation of the six-helix bundle formation and the N-terminal cap structure in HA2 of AIV.
2.To establish a cell-based pharmacological model for compounds screening by using the H5N1 pseudotyped viral system.The plasmids encoding the AIV Envs HA and NA and the pNL43Luc.RE-(with a luciferase reporter gene and a HIV-1 core gene) were transfected into 293T cells for expression of HA and NA on pseudotyped virus containing the HIV-1 core.This system would be used for screening of anti-AIV compounds.
3.To develop in vitro H5N1 AIV infection model using MDCK cells and in vivo H5N1 AIV infection model using the Balb/c mice for evaluation of the in vitro and in vivo efficacy of anti-AIV compounds.
4.To establish an XTT assay and a mouse model for evaluating the in vitro and in vivo toxicity,respectively,of the selected anti-AIV compounds.
5.To establish an HPLC method for studying the pharmacokinetics of ARC-36 in mice.
RESULTS:
1.The results from FLISA,natural fluorescence gel electrophoresis,SE-HPLC analysis showed that there were no detectable interactions between N29 and C19 and between N8 and C8 peptides derived from the HA2 domain of H5N1 AIV.
2.The HA-NA/HIV pseudovirus could infect 293T cells and MDCK cells,and ARC-36 could inhibit the AIV pseudovirus infection with IC_(50) value of 4.00±0.38μM.
3.ARC-36 also inhibited live H5N1 virus infection of MDCK cells with IC50 of 27.03±2.54μM.It had now toxicity to MDCK cells with CC_(50) of 525.4±63.22μM.Intranasal administration of ARC-36 before the intranasal infection protected mice from H5N1 AIV challenge,with the average survival time of 8.8 days,while 7.4 days for the control normal mice,a significant differences(p＜0.01).
4.As analyszed by HPLC(A phase:100%methanol,B phase:0.5%glacial acetic acid),the retention time of ARC-36 was 6.3 min with a standard curve equation of Y= 5354.2X+7106.5,r = 0.9993(n = 5).The Ct curve is two-compartment model after intraperitoneal injection of ARC-36.The elimination half-life(t1/2)_βis 31.6min.The LD50 of ARC-36 by intraperitoneal injection is 149.2 mg·kg~(-1) (7.5mg·ml~(-1),211.8μM),and is about 50-fold of the IC_(50) value of ARC-36 determed in pseudovirus assay(4.00±0.38μM).
CONCLUSION:
1.Because of no detectable interactions between N29 and C19 and between N8 and C8,we failed to develop these peptides-based high-throughput screening methods for screening of the compounds that inhibit formation of six-helix bundle and the N-terminal cap structure in HA2 of H5N1 AIV.
2.I successfully developed the HA-NA/HIV pseudovirus infection system using 293T cells and MDCK cells for screening of anti-AIV compounds and using this system,we found that ARC-36 was effective in inhibiting AIV infection,but was infective when it was added 24 hours post infection.
3.I established the Balb/c mouse AIV challenge model and MDCK cell H5N1 AIV infection model for evaluating the in vivo and in vitro efficacy of the selected anti-AIV compounds.We confirmed that ARC-36 is effective in inhibiting H5N1 AIVinfection in MDCK cells.Intranasal administration of ARC-36 before the intranasal infection resulted in prolonged average survival time of mice.
4.I established the HPLC method for quantitative analysis of ARC-36 level in vivo. I found that he Ct curve is two-compartment model after intraperitoneal injection of ARC-36,and the elimination half-life(t1/2)_βis 31.6 min,owing to the quick metabolism.The LD50 of ARC-36 by intraperitoneal injection is 50-fold of the IC50 value determined in pseudovirus assay,suggesting that ARC-36 is relatively safe.
PARTⅡ.Down-regulation of antigen-specific T cell activation by a peptide HIV-1 fusion inhibitor via interaction with T cell receptor
OBJECTIVE:
To study the effect of VIR576 on antigen-specific T cell(Ab2 cells,CD4~+ CD25T cells) activation and non-antigen-specific T cell activation;To analyze the membrane distribution of VIR576 in T cell using the fluorescent confocal microscope and the colocalization of VIR576 with TCR;To study the interaction of VIR576 and the synthetic peptide derived from TCR-TMD,the CP sequence(aa 253-261),using hemolysis test,hemolysis inhibition assay,FRET,FLIA,and competitive FLISA.
METHODS:
1.To investigate the potential effect of FP16,VIR576,and the mixture of FP16 and VIR576 on the proliferation of A2b cells in response to MOG 35-55 at 10μg/ml, to test the effect of FP16,VIR576 and their mixture on proliferation of splenocytes of OVA-specific DO 11.10 Tg mice in response to chicken OVA,and to test the proliferation activity of non-stimulated and Con A-stimulated splenocytes from BALB/c.
2.To establish TCR-TMD-mediated hemolysis assay,and using this assay,to study the effect of VIR576 on the hemolytic activity of TCR-TMD.
3.To develop a simple and rapid fluorescence-based binding assay using Rhodamine(Rho)-conjugated VIR576 to evaluate the potential interaction between VIR576 and TCR-TMD,to conduct a competitive assay to confirm the specific interaction between TCR-TMD and Rho-VIR576 using unlabeled-VIR576 and VIRIP scramble peptide,and to identify the critical binding site of VIR576 in TCR-TMD.
4.4.To study whether VIR576 could colocalize with CD4 molecule in the CD4~+ T cell membrane to interact with TCR.
RESULTS:
1.VIR576 alone suppressed the proliferation of A2b T cells in responses to MOG 35-55,and it could also block FP16-mediated inhibition of A2b cell activation (p＜0.05).VIR576 itself also inhibited splenocytes proliferation in responses to OVA,and it could block FP16-mediated inhibition of splenocytes activation (p＜0.05).Both splenocytes and CD4~+CD25~-T cells of DO11.10 mice showed a dose-dependent strong proliferative in response to the stimulation by OVA,while the addition of VIR576 resulted in significant inhibition of proliferation of splenocytes and the primary CD4~+CD25~- T cells of DO11.10 mice.The proliferation of non-stimulated and Con A-stimulated splenocytes was not affected by addition of VIR576 at the concentration as high as 50μg/ml. Consistently,VIR576 had no significant effect on proliferation of DO11.10 mouse splenocytes in the absence or presence of Con A or anti-CD3 antibody.
2.VIR576 interacted with CP since incubation of human erythrocytes with TCR-TMD resulted in significant hemolysis in a dose-dependant manner with IC_(50) of 1.23±0.16μM,whereas the VIRIP scramble peptide had no significant inhibition on TCR-TMD-mediated hemolysis(p＜0.05).Rho-VIR576 bound to TCR-TMD in a dose-dependant manner,resulting in a significant increase of the fluorescence intensity.A significant decrease in fluorescence intensity when mixture of unlabeled-VIR576 and Rho-VIR576 were added to the immobilized TCR-TMD,while the unlabeled VIRIP scramble peptide had no effect on the binding of Rho-VIR576 to TCR-TMD(p＜0.05).When Rho-VIR576(final concentration 0.04-0.12μM) was added to a mixture of NBD-CP(0.04μM) and PC LUV liposome(100μM),a dose-dependent quenching of the donor's emission,which is consistent with energy transfer,was observed.
3.Rho-VIR576 did not bind to the unstimulated lymphocytes,but strongly interacted with the CD4~+ T cells stimulated with anti-CD3 antibody.Only a part of activated T cell membrane was stained by Alexa-labeled anti-CD4 antibody.
CONCLUSION:
1.VIR576 was effective in suppressing the antigen-specific T cell activation,but has no effect on non-specific T cell proliferation.
2.VIR576 had the ability to downregulate the antigen-specific T cell activation by interaction with TCR transmembrane domain.
3.Rho-VIR576 strongly interacted with the CD4~+ T cells stimulated with anti-CD3 antibody.
引文
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[1]Holmes EC.Virology: 1918 and all that [J].Science , 2004, 303 (5665): 1787-1788.
[2]Layne SP, Beugelsdijk TJ, Patel CK, et al.A global lab against influenza [J].Science, 2001, 293(5536): 1729.
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