酸酐修饰人血清白蛋白的抗病毒活性及安全性研究
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摘要
自1981年第一例HIV/AIDS感染病例在美国被报道以来,艾滋病迅速地在全世界范围内蔓延。理论上,HIV感染最好的预防手段是研发艾滋病疫苗。然而,目前仍没有一个艾滋病疫苗被临床试验证明为有效。目前,治疗HIV主要手段是使用各种抗病毒药物。目前临床批准的抗艾滋病药物已经有29种,但绝大部分针对病毒编码的两种酶,即逆转录酶和蛋白酶。尽管有这么多的抗HIV药物,但它们仍然不能清除HIV,只能抑制病毒的复制,将病毒载量降低,定程度地恢复患者的免疫功能,延长患者的生命。直到2003年,第一个HIV进入抑制剂衍生于病毒包膜蛋白gp41的多肽恩夫韦特(T-20)被FDA批准进入临床,开创了病毒进入抑制剂作为抗病毒药物的新时代。HIV进入抑制剂虽然能从源头上阻止病毒的入侵,但其仅能特异性地抑制HIV-1,对HIV-2没有抑制活性,同时多肽类药物价格昂贵,也限制了它们的临床应用。另一棘手的问题是HIV病毒变异性极高,极易产生耐药株,从而使现有的许多抗病毒药物失活。联合3种或4种抗病毒药物(通常包含一种蛋白酶抑制剂)的高活性抗逆转录病毒联合疗法(Highly Active Anti-Retroviral Treatment, HAART)目前已成为治疗HIV感染的标准方法,其可以显著降低HIV感染患者的死亡率和发病率。HAART通常被称为“鸡尾酒疗法”(Cocktail therapy)。不同作用机制的抗病毒药物之间的相互作用及合理搭配使HAART的效果比单一用药有优势,能有效地降低用药剂量,减少不良反应,提高抗病毒谱,更为重要的是能减少耐药病毒株的产生。因此,急需寻找新的来源的药物抑制HIV的广泛传播。
人血清白蛋白(HSA)是人血浆中的最为主要的蛋白质,其具有许多重要的生理学和药理学功能,目前已经成为基础及临床研究最广泛的目标蛋白之一。尤其是对HSA进行适当的化学修饰,利用化学修饰产物作为理想的治疗分子进行研发,已经成为生命科学中重要的研究手段之一。HSA其非糖基化的单链多肽包含585个氨基酸,相对分子量为66.5kD,主要是由18种氨基酸残基组成,其中碱性氨基酸赖氨酸有59个,精氨酸则有27个。由于酸酐是强酸性物质,而蛋白质末端含有很多碱性的氨基酸,因此两者发生化学反应时,其蛋白质末端的碱性氨基酸残基必然会被酸酐所修饰。如果将蛋白末端的这些碱性氨基酸,选用适合的酸酐进行化学修饰,将得到一类新的物质,即酸酐修饰的HSA。1995年,美国纽约血液中心的Neurath AR和姜世勃等人的研究发现3-羟基-邻苯二甲酸酐(3-hydroxyphthalic anhydride,3-HP)修饰的p-乳球蛋白((3-lactoglobulin,β-LG),即3-HP-β-LG,可特异性抑制HIV的感染。未经修饰的β-乳球蛋白及3-HP本身都无抑制HIV的活性,说明酸酐修饰使无活性的蛋白产生了抗HIV活性。然而由于时逢全球疯牛病爆发,使得来源于牛奶蛋白的制剂3HP-β-LG的研发受到了极大的影响,将其研发的试验研究被迫终止。
HSA是人血液中最为主要的一种蛋白质,其含量占血浆中总蛋白含量的60%以上,因此含量丰富。而且结构较为稳定,在人血管和血管外循环中分布较广,半衰期长达19d,无明显的免疫原性,是血液中重要的物质输送载体,因此将其做为抗病毒药物研发具有一定的优势。因此,本文采用类似的方法,选用人血清白蛋白作为被修饰蛋白,来研究酸酐修饰人血清白蛋白特性和评价其抗病毒活性及其可能的临床应用。
1.酸酐修饰人血清白蛋白HP-HSA的制备和条件优化
本文参考文献方法,选用3-羟基-邻苯二甲酸酐HP来修饰人血清白蛋白HSA,为优化HP-HSA的制备条件,我们选用不同浓度的HP (20mM、40mM及60mM)和不同的pH值反应体系(pH7.0、8.0及9.0),合成了的一系列不同的HP-HSA,并评价这些HP-HSA的抗两种HIV-1实验室病毒株(X4及R5型)的活性。同时,我们还通过检测不同条件下合成的HP-HSA期蛋白末端碱性氨基酸(主要包括赖氨酸及精氨酸)残基被HP修饰比率,比较HP-HSA的抗病毒活性与蛋白末端碱性氨基酸被修饰比率之间的相关性。
结果发现,HSA蛋白末端的碱性氨基酸残基大部分能被HP所修饰,其蛋白的化学结构发生了变化,从而将无活性的HSA转变成具有较强抗HIV-1活性的病毒抑制剂HP-HSA。HP-HSA中赖氨酸和精氨酸残基的被修饰的比率越高,其抗抗HIV-1X4型(HIV-1ⅢB)及R5型(HIV-1Bal)的病毒株的作用也相应的越强,说明蛋白末端碱性氨基酸被修饰的比率与HP-HSA抗病毒活性密切呈正相关,修饰蛋白末端的碱性氨基酸残基被相应酸酐修饰的比率与酸酐修饰蛋白的抗病毒活性密切相关。通过比较分析确定,HP-HSA化学修饰的最优合成条件为,酸酐HP终浓度60mM,pH值为9.0,在这一条件下,HP-HSA的蛋白末端赖氨酸被修饰的比率为98.17%,而精氨酸被修饰的比率为91.67%。而在这一条件下,合成的HP-HSA的抗HIV-1IIIB(X4型)和BaL(R5型)病毒的IC50值分别是0.851nM和1.454nM。
2. HP-HSA抗病毒活性研究
作为一个理想的防治性传播性疾病的抗病毒制剂,首先要保证其具有广谱性和有效性,即能够高效抑制绝大部分与性传播疾病有关的病原体的感染。与3HP-β-LG相类似,酸酐修饰蛋白HP-HSA也具有广谱高效的抗病毒活性,其能抑制多种HIV-1实验室病毒株包括HIV-1X4型和R5型,IC50在低纳摩尔水平。
另外,HP-HSA也能有效抑制HIV-2感染,意味着HP-HSA也能被用于HIV-2病毒广泛流行的西非等国家。同时HP-HSA还能抑制SIV的感染[4]。由于SIV病毒能够感染恒河猴,因此利用SIV病毒阴道攻击恒河猴的动物模型,可先期评价HP-HSA在动物体内的抗病毒活性。
目前大多数的抗病毒药物,尤其是临床上应用最为广泛的逆转录酶抑制剂临床应用的局限性最主要的是由于HIV病毒耐药株的产生。HIV病毒需寄生于宿主细胞内,利用宿主细胞的代谢系统生存、增殖,并保持极强的生命力。HIV病毒在宿主细胞内不断复制,但是其复制的错配率极高且极易产生高变异性,这也是目前理想抗HIV病毒药物研发缓慢的主要原因之一。本文研究发现,HP-HSA对几类常见的抗病毒药物包括核苷类逆转录酶抑制剂AZT,非核苷逆转录酶抑制剂A17及HIV进入抑制剂T20的耐药病毒株,均具有非常好的抑制作用,其IC50值均在低纳摩尔水平,与抗HIV-1实验室病毒分离株的抗性相当。这弥补了这些目前临床应用的主要的抗病毒药物的不足,从而使其具备了被进一步开发的前景。如果将这类蛋白类药物与抗病毒药物联合应用,可能也将成为抗病毒药物研究的一个新的方向。
如前所述,生殖器单纯疱疹病毒II型(Herpes Simplex Virus, HSV-2)是另一类导致性传播性疾病STD的主要病原体,约75%生殖器疱疹病例是由HSV-2引起的,它是引发生殖器疱疹和溃疡最主要的原因,是一个世界性的公共卫生问题。值得注意的是,其引发的生殖器疱疹据报道与HIV感染的关系极为密切,研究发现患有生殖器疱疹的病人更容易感染HIV,其HIV感染率可增加2-4倍。因此,有效控制HSV-2感染对减少HIV的流行具有直接的作用。如果抗病毒药物在具有抗HIV活性的同时,具有抑制HSV-2病毒的作用,则将大大增加该抗病毒药物的抗HIV活性,从而成为高效双功能的抗病毒制剂。目前,临床应用的大部分抗HIV药物均为窄谱抗病毒药物,其无或者对HSV-2的抑制作用不强。本文我们研究发现,酸酐修饰人血清白蛋白HP-HSA对HSV-2病毒有明显抑制作用。
3. HP-HSA抗病毒作用机制研究
HIV病毒进入CD+4靶细胞一般是在1h内完成的。如果一种药物为HIV进入抑制剂,其必须在病毒进入靶细胞前与病毒或靶细胞接触。因此,我们选用time-of-addition的分析方式,在病毒感染靶细胞的不同阶段加入酸酐修饰蛋白,以观察HP-HSA到底作用在HIV感染的哪个环节。实验结果发现,当HP-HSA在病毒感染2h后加入时,其抗HIV-lHIB和抗HIV-1BaL病毒活性完全消失,其作用与FDA批准的三种HIV进入抑制剂如T20,AMD3100(CXCR4辅助受体抑制剂)及Maraviric (CCR5辅助受体抑制剂)药物的失活时间相似,而对照药物逆转录酶抑制剂AZT,即使在病毒感染靶细胞8h后加入,仍然能保持较高的抗HIV-1活性。这些结果说明HP-HSA是一类特异性的HIV进入抑制剂,对HIV病毒复制的其他环节无明显作用。
由于阻断HIV病毒生命周期的第一个阶段,因此靶向HIV进入阶段的抗病毒药物均具有明显的优势。研究表明,游离病毒和已感染HIV的靶细胞都能感染新的靶细胞,而后者在HIV的感染中占主要地位。在HIV的感染过程中,首先是HIV包膜与靶细胞膜发生融合,HIV基因组进入靶细胞。HIV进入靶细胞是由病毒表面的包膜糖蛋白介导的。因此,阻止HIV病毒的进入,关键是阻止HIV Env诱导的细胞-细胞融合及病毒-细胞融合过程。通过细胞-细胞融合、病毒-细胞融合及细胞间的传播等实验结果分析可知,HP-HSA的确作用于病毒的进入阶段,是一类HIV进入／融合抑制剂。
HIV通过三个步骤进入宿主细胞：1)HIV颗粒通过其包膜上的糖蛋白gp120与靶细胞表面的CD4受体粘附；2)gp120发生构象改变以适应与靶细胞上的辅助受体CCR5或CXCR4结合；3)HIV跨膜蛋白亚基gp41介导病毒包膜与靶细胞膜发生融合。那么HP-HSA到底作用于HIV进入的哪个环节,则成了我们下一步关心的问题,因此我们对HP-HSA的作用靶点进行研究。
HIV进入靶细胞的第一步就是HIV病毒颗粒通过其包膜糖蛋白gp120与靶细胞表面的CD4受体粘附,因此,我们首先检测HP-HSA是否抑制gp120和可溶性CD4分子(sCD4)的体外结合。结果发现,HP-HSA能抑制来源于HIV-1HIB和HIV-1BaL的gp120与sCD4的结合,而未修饰HSA则无抑制作用。这些结果表明,HP-HSA可能是通过抑制病毒包膜糖蛋白gp120结合到靶细胞上的CD4受体而产生抑制作用的。ELISA和流式分析结果进一步表明,HP-HSA可通过与病毒包膜糖蛋白gp120和靶细胞上的CD4受体结合,阻止HIV-1的gp120与CD4两者的相互作用,从而产生抑制病毒与细胞膜融合的过程。HSA蛋白表面正电荷的精氨酸和赖氨酸残基被酸酐修饰之后,其表面聚集着大量的负电荷侧链,这些负电荷侧链能与gp120和CD4分子结合,从而产生抗病毒作用。
4. HP-HSA的局部用药安全性研究
理想的抗病毒药物除了具备上述价廉和高效等条件外,更为重要的一点即为其安全性。抗病毒药物由于可能每日被多次使用,且长期甚至终生服用,因此必须不会导致组织损伤,不会产生过敏反应,不会改变阴道内酸性环境,不会造成阴道内菌群失调,同时,应无全身性毒副反应。在体液中仍能保持较好的抗病毒活性。
阴道粘膜是多层鳞状上皮细胞,它们组成了防御外来病毒的一道物理屏障。因此,任何能破坏上皮组织完整性的因素,譬如物理损伤、性传播病原体的感染、局部溃疡及炎症、激素水平失衡、阴道正常菌群失调、以及不当使用阴道洗液等,都可能提高HIV的感染率。因此,药物对阴道上皮细胞的毒性是其能否用于临床的关键。我们首先评价了HP-HSA对3种T淋巴细胞和3种正常人阴道及子宫颈上皮细胞的体外毒性,结果显示,HP-HSA对测试的6种病毒作用T淋巴靶细胞及正常人阴道及子宫颈组织细胞的细胞毒性作用很低,其CC50的值远远大于其抑制HIV-1HIB病毒的IC50值,这意味着HP-HSA体外基本无毒。
阴道乳酸菌是内源性的杀微生物剂,它能通过产生乳酸和过氧化氢来发挥效能。流行病学研究表明产过氧化氢乳酸菌能有效降低HIV的感染率。其原因推测是间接的,产过氧化氢乳酸菌能降低细菌性阴道炎的发病率,而后者导致的阴道上皮组织损伤是HIV传染的一个主要影响因素。因此,我们评估了HP-HSA对17株正常人阴道乳酸杆菌生长的影响。药敏实验结果发现,较高浓度的HP-HSA对阴道正常乳酸杆菌的生长无明显影响,而阳性对照药物氨苄青霉素对阴道乳酸杆菌各株均有明显抑制作用,其MIC为0.313～1.25
前面研究证实,HP-HSA的作用靶点之一为T淋巴细胞表面的CD4受体,而CD4+T淋巴细胞是人免疫系统中极为重要的一类细胞,对人正常免疫功能起着至关重要的作用[8]。那么,HP-HSA作为杀微生物剂用于机体后,是否会对T淋巴细胞的正常的功能产生影响?首先,我们观察了HP-HSA是否对正常人PBMCs增殖的影响,同时观察了HP-HSA对PHA刺激的PBMCs增殖的反应,结果证明即使在浓度为100μM时,HP-HSA对正常和PHA刺激的PBMCs的生长增殖均无明显的影响。随后,我们评估了HP-HSA对正常和PHA刺激的PBMCs分泌细胞因子IFN-γ功能的影响,结果同样证明HP-HSA对正常和PHA刺激PBMCs的免疫功能无影响。这些结果初步证明,HP-HSA对CD4+T细胞的功能,尤其是处于体内循环状态的T细胞不会产生有害的影响。但是,我们也不能排除长期应用阻断CD4受体的杀微生物剂可能会抑制阴道粘膜中的CD4+T细胞的免疫功能,这也正是我们下一步准备进行的安全性实验,评估HP-HSA对家兔阴道粘膜免疫系统的影响。
性传播是目前HIV传播的主要途径,因此人体精液和宫颈阴道液对抗HIV药物活性影响的评估就显得至关重要了。一般来说,理想的抗病毒药物还应该在存在大量体液时,仍能保持较强的抗病毒活性。有文献报道,精液的存在会降低一些候选杀微生物剂的活性。本文结果可见,人体正常精液和阴道模拟液对HP-HSA的抗病毒活性并无显著影响。(五)小结
HP-HSA为水溶性蛋白,其无味,高浓度是呈淡黄色,制备简单、快速、可重现,且其体外毒性、免疫功能和稳定性评估证明其安全稳定,具有被开发成抗病毒药物的潜能。HP-HSA的优势：1)具广谱抗病毒活性,能对抗HIV-1,HIV-2, SIV和HSV的感染；2)能有效对抗各类药物的耐受株,包括核苷逆转录酶抑制剂耐药株、非核苷逆转录酶抑制剂耐药株及T20耐药株；3)HP-HSA为来源于人的大分子蛋白类药物,其生物半衰期长；4)HSA来源于人类,其抗原性较低,不易诱导不利的免疫反应。然而,HP-HSA作为蛋白类药物可能存在价格较高、给药方式较难选择等缺陷。将两类不同作用机制或不同作用靶点的新型抗病毒药物与临床应用的抗病毒药物联合应用,将有效避免各自的优缺点,取长补短,发挥各自的优势,从而产生协同作用效果。
Since the first cases of HIV/AIDS were reported in1981in the United States, AIDS is spreading worldwide rapidly. Theoretically, the best method to preventing HIV infection is to develop an HIV vaccine. However, there is still no effective HIV vaccine to be used in human beings. Currently, the major treatment for preventing HIV infection is the antiviral drugs. Until now,29antiviral drugs with different mechanism of actions have been approved by Food and Drug Administration (FDA). Most of them are belongs to two classes. One is reverse transcriptase inhibitor and the other is protease inhibitor. In spite of so many kinds of anti-HIV drugs used in patients, HIV can not be killed clearly. Those drugs can only suppress viral replication, reduce viral load and partly restore a patient's immune function and prolong the lives of patients. In2003, the first HIV entry inhibitor, Enfuweite (T-20), was approved by FDA, which is a peptide derived from HIV envelope protein gp41. HIV entry inhibitor can prevent the virus invasion at the first stage. However, it only specifically inhibits HIV-1infection but not inhibiting HIV-2infection. Otherwise, peptide drugs have expensive price, which also limit the clinical application. Another thorny issue is the high mutation of HIV virus. It is extremely easy to produce drug-resistant strains, which can reduce the antiviral activities of most of antiviral drugs. The highly active antiretroviral combination therapy (Highly Active Anti-Retroviral Treatment, HAART) is always combining with three or four kinds of antiviral drugs (typically containing a protease inhibitor). It has become a standard method of treating HIV infection, which is significantly reduced the mortality and morbidity of HIV-infected patients. HAART is also called as "cocktail therapy". A combination of different mechanisms of antiviral drugs may have the following advantages:(i) maximizing antiviral efficacy because of the potent synergistic activity against HIV-1laboratory-adapted both X4and R5strains,(ii) minimizing toxic effects and high cost due to combining dose reduction, and (iii) fighting against diverse anti-HIV-1drug mutations or delaying the emergence of HIV-1resistance by using agents with different mechanisms. Therefore, the devlepment of effective, safe and affordable new antiviral drugs to preventing HIV transmission is urgently needed.
Human serum albumin (HSA) is the most common protein in human plasma. It has a lot of important functions of physiology and pharmacology. Now HSA has become one of the most extensive target proteins in basic and clinical research, especially on appropriate chemical modification. Development a chemically modified product as an ideal therapeutic molecule has become one of the important means of research in life sciences. The non-glycosylated single polypeptide chain of HSA contains585amino acids and a relative molecular weight was66.5kD. It is mainly composed of18kinds of amino acid residues, in which contains59lysine and27arginine. Since the anhydride is a strongly acidic substance, the protein containing many alkaline amino acids can be modified by the anhydride. After chemical modification, an anhydride-modfied protein will be produced.
In1995, Neurath AR and Shibo Jiang in our group of New York Blood Center demonstrated that bovine β-lactoglobulin, a protein present in milk and whey, modified by3-hydroxyphthalic anhydride (3HP-β-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1and HSV-2). Nether non-modified B-lactoglobulin nor3-hydroxyphthalic anhydride had the anti-HIV activity. It means the chemical modification converted commonly available proteins into potent antiviral compounds.3HP-β-LG is highly stable in aqueous solution for long-term storage at room temperature and elevated temperatures. However, the outbreak of bovine spongiform encephalopathy (BSE) in Europe raised a safety concern with regard to developing bovine proteins for medical use, resulting in discontinuation of further development of3HP-β-LG as a microbicide.
HS A is the main protein in human blood, and it is composed of more than60%of the total proteins in the plasma. The structure of HSA is stable and it widely distributed in the human vascular and extravascular circulation. The half life of HSA is up to19days. Because of its human-derived protein, it is no obvious immunogenicity. Therefore, the anhydride modified HSA has the superiority to be developed an ideal antiviral drug in future. Therefore, we sought to replace bovine proteins with chemically modified HSA using the same method and the same conditions as3HP-β-LG Its antiviral activity and its possible clinical applications will be evaluated in the present study.
Optimization of the conditions for chemical modification of HP-HSA
HP-HSA was prepared according to the procedures previously described. For Optimization of the conditions for chemical modification of HP-HSA, we prepared HP-HSA under six different sets of conditions and evaluated the correlation between the anti-HIV-1activities (both X4and R5) and the percentage of lysine and arginine residues in HP-modified and-unmodified HSA to optimize the conditions for production of HP-HSA. Results showed that, with the increased concentration of HP (20,40and60mM, pH9.0) and the pH of the reaction system (7.0,8.0and9.0, concentration of HP is60mM), a higher percentage of lysine and arginine residues was modified, leading, in turn, to stronger anti-HIV-1activity on both IIIB (X4strain) and BaL (R5strain) viruses. Based on these results, we selected as the optimal condition (60mM of HP at pH9.0) for preparation of the HP-HSA. Under such condition, the EC50s of HP-HSA are about0.851and1.454nM on ⅢIB and BaL viruses, with98.17%and91.67%of the lysine and arginine residues modified by HP, respectively (Table1).
Determination of the antiviral activities of HP-HSA
It is desirable that a topical antiviral drug should be a broad-spectrum and effective antiviral agent. By ELISA, HP-HSA effectively inhibited infection by laboratory-adapted HIV-1strains, including X4and R5viruses, with IC50in the nM range.
HP-HSA is also effective in inhibiting HIV-2infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2is prominent. Our studies also showed that HP-HSA could potently inhibit infection by SIV. Results showed that HP-HSA strongly inhibited the infection by HIV-2ROD, and SIVmac25132H viruses, with low EC50s in the range of8.582to65.769nM Since SIV can be used for infection of rhesus macaques, HP-HSA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SIV infection through vaginal challenge..
Still, one of the most prominent problems facing future antiviral drugs is the development of resistant HIV-1strains against reverse transcriptase inhibitors. Our evidence demonstrated that HP-HSA retained high activities against different resistant HIV-1variants, including NRTI-resistant Zidovudine-R, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant A17-R, and HIV-1entry inhibitor-resistant NL4-3(36G)V38A and NL4-3(36G)V38E/N42S (Table1). Those results indicate that HP-HSA is capable of preventing the sexual transmission of HIV-1strains that are resistant to the currently used antiretroviral therapeutics.
As we described above, herpes simplex virus-2(HSV-2) produces most genital herpes, which is primarily associated with HIV transmission. Clinical data indicate that patients with prevalent HSV-2infection have a two to four-fold higher risk of HIV acquisition than HSV-2uninfected population. Therefore, we investigated the efficacy of HP-HSA against HSV-2in vitro in this study. Results showed that HP-HSA also has very low inhibitory activity on HSV-2and the ECsos value was34.514nM. The negative control protein, unmodified HSA even at concentrations up to10.000uM, had no inhibitory activity against any of the above viruses (data not shown). Cell viability analysis results showed that HP-HSA at the concentration of the effective concentration for100%inhibition (EC100) and lower than EC100had no cytotoxicity to virus target cells (Vero cells).
Evaluation the possible mechanism of HP-HSA on HIV-1infection.
As described before, HP-HSA exhibited significantly decreased inhibitory activity when it was added to target cells0.5-2h post-HIV-1infection by time of addition assay.6While such results indicated that HP-HSA was an HIV-1entry/fusion inhibitor, the potential targets of HP-HSA remained unknown. Therefore, to systematically search for these targets, we examined the effect of HP-HSA on the early steps of the HIV-1replication cycle, i.e., HIV-1fusion/entry, using cell-cell fusion and virus-cell fusion assays.
HIV-1Env--mediated cell-cell fusion was detected using fusion of noninfectious CHO-WT cells with MT-2cells or infectious HIV-1ⅢB-infected H9cells (H9/HIV-1Ⅲb cells) with MT-2cells, respectively. In noninfectious analysis system, the giant syncytia were observed by an inverted microscope. The size of syncytium is usually twice or more times than the size of normal cells. In infectious analysis system, the fluorescence diffuses were observed by an inverted fluorescence microscope. The numbers of the formation of giant syncytia and fluorescence diffuses in cell-cell fusion assay were counted by choosing four fields randomly under an inverted microscope. HP-HSA could significantly decrease the formation of giant syncytia and fluorescence diffuses in a dose-dependent manner. The percentage of inhibition of cell-cell fusion of HP-HSA and HSA were calculated according to the formula described above. Results showed that HP-HSA inhibited both kinds of HIV-1Env-mediated cell-cell fusion, with EC50about27.82nM in a noninfectious system and7.45nM in an infectious system, respectively. In the above-described assays, unmodified HSA demonstrated no ability to inhibit cell-cell fusion.
A single-round entry assay was chosen to detect the inhibition of HP-HSA on direct virus-cell fusion by using pseudotyped viruses expressing HIV-1HXB2(X4strain) and SF162(R5strain) Env. Similar results were observed in this assay. HP-HSA inhibited infection by both X4and R5Env-pseudotyped viruses, with EC50about1.51and1.54nM, respectively. Vesicular stomatitis virus-G (VSV-G) pseudovirus was produced by cotransfecting VSV envelope G-protein plasmid and pNL4-3E-R-Luc plasmid. Therefore, VSV-G pseudovirus could be used to evaluate the specific effects of HP-HSA on HIV-1envelope proteins. Results showed that VSV-G pseudovirus infection could not be inhibited by HP-HSA, even at the concentration as high as1.25Ｌ.M
Cell-to-cell transmission is the major route for the spread of HIV-1. Here, we detected the inhibition of HP-HSA on the transmission of cell-associated HIV-1virions from PBMCs to CEMxl745.25M7cells. HP-HSA blocked the cell-cell HIV-lBaL transmission between those two kinds of virus target cells, which suggests that it could prevent the transmission of cell-associated HIV-BaL isolates. The EC50of cell-cell transmission was31.01nM. Unmodified HSA had no inhibitory activity on cell-cell transmission.
All of these results indicate that HP-HSA can inhibit HIV-1infection by blocking the virus entry step. HIV-1entry into a CD4+T cell is mediated by viral envelopes, which comprise the surface subunit gpl20and transmembrane subunit gp41. Similar to HP-OVA and ML-OVA, one of the probable mechanisms of HP-HSA might be interference with the viral envelope glycoprotein or the receptor on the target cell membrane.
Evaluation the safty of HP-HSA
general, HIV transmission during sexual intercourse should be occurred in presence of normal human body fluids, such as seminal and vaginal secretions. The antiviral activities of an ideal antiviral drug should be retained in the presence of those human body fluids. Here, we determined the potential effect of SP and VFS on the anti-HIV activity of HP-HSA. Results showed that neither SP nor VFS had significant effect on the inhibitory activity of HP-HSA against infection by both HIV-1X4(ⅢB) and R5(BaL) strains. The EC50values of HP-HSA on HIV-1ⅢB strains in the presence of PBS, SP or VFS were1.605nM,1.532nM or1.408nM, respectively. Moreover, the EC50values of HP-HSA for inhibiting HIV-1BaL infection in the presence of SP and VFS were4.997nM and7.564nM, respectively, whereas that of PBS control is6.477nM.
Lactobacilli are the predominant organisms in the healthy vagina of women, which maintain a normal vaginal microbiota and form a natural physical barrier against other bacteria or viruses. Here, we chose17strains of normal human vaginal lactobacilli and evaluated the effects of HP-HSA on the proliferation of those vaginal lactobacilli. Results showed that HP-HSA had not produced significant inhibitory effect on17strains of vaginal lactobacilli (MIO15.000μM). The MICs of positive control Ampicillin on different strains were from0.841μM to6.730μM.
Normal CD4+T lymphocytes in human beings are essential in the immune system, such as helping antigen presenting cell expresses an antigen on MHC class II through a combination of cell-to-cell interactions or through cytokines.32As we described above, since HP-HSA can bind to cellular CD4receptor, a concern is raised about the function of CD4+T lymphocytes in the presence of HP-HSA.33In our previous studies, we confirmed that HP-HSA had no significant effects on the proliferation of T lymphocytes and no obvious effects on the production of IFN-y in either human PBMCs or PHA-stimulated PBMCs. One advantage of HP-HSA is its origin from human sera, which may reduce any deleterious effects on the function of CD4+T cells, especially for those circulating in the blood stream. Nonetheless, the long-term use of CD4blockers could still suppress the function of CD4+T cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-HSA on the mucosal immune system is warranted.
Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-HSA is broad-spectrum HIV entry/fusion inhibitors through blocking viral entry. By its broad antiviral potency, resistance to trypsin hydrolysis, easy preparation, low production costs, wide availability and absence of carcinogenic phthalic group, HP-HSA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-HSA may be used in combination with an NNRTI-based microbicide for preventing sexual transmission of HIV because the combination may have synergistic antiviral activity against a broad spectrum of HIV-1strains and reduce the potential toxic effects.These findings might be helpful to looking for a novel strategy for treatment of HIV/AIDS and might provide a rational basis for testing of microbicide combinations in vivo.
人血清白蛋白(HSA)是人血浆中的最为主要的蛋白质,其具有许多重要的生理学和药理学功能,目前已经成为基础及临床研究最广泛的目标蛋白之一。尤其是对HSA进行适当的化学修饰,利用化学修饰产物作为理想的治疗分子进行研发,已经成为生命科学中重要的研究手段之一。HSA其非糖基化的单链多肽包含585个氨基酸,相对分子量为66.5kD,主要是由18种氨基酸残基组成,其中碱性氨基酸赖氨酸有59个,精氨酸则有27个。由于酸酐是强酸性物质,而蛋白质末端含有很多碱性的氨基酸,因此两者发生化学反应时,其蛋白质末端的碱性氨基酸残基必然会被酸酐所修饰。如果将蛋白末端的这些碱性氨基酸,选用适合的酸酐进行化学修饰,将得到一类新的物质,即酸酐修饰的HSA。1995年,美国纽约血液中心的Neurath AR和姜世勃等人的研究发现3-羟基-邻苯二甲酸酐(3-hydroxyphthalic anhydride,3-HP)修饰的p-乳球蛋白((3-lactoglobulin,β-LG),即3-HP-β-LG,可特异性抑制HIV的感染。未经修饰的β-乳球蛋白及3-HP本身都无抑制HIV的活性,说明酸酐修饰使无活性的蛋白产生了抗HIV活性。然而由于时逢全球疯牛病爆发,使得来源于牛奶蛋白的制剂3HP-β-LG的研发受到了极大的影响,将其研发的试验研究被迫终止。
HSA是人血液中最为主要的一种蛋白质,其含量占血浆中总蛋白含量的60%以上,因此含量丰富。而且结构较为稳定,在人血管和血管外循环中分布较广,半衰期长达19d,无明显的免疫原性,是血液中重要的物质输送载体,因此将其做为抗病毒药物研发具有一定的优势。因此,本文采用类似的方法,选用人血清白蛋白作为被修饰蛋白,来研究酸酐修饰人血清白蛋白特性和评价其抗病毒活性及其可能的临床应用。
1.酸酐修饰人血清白蛋白HP-HSA的制备和条件优化
本文参考文献方法,选用3-羟基-邻苯二甲酸酐HP来修饰人血清白蛋白HSA,为优化HP-HSA的制备条件,我们选用不同浓度的HP (20mM、40mM及60mM)和不同的pH值反应体系(pH7.0、8.0及9.0),合成了的一系列不同的HP-HSA,并评价这些HP-HSA的抗两种HIV-1实验室病毒株(X4及R5型)的活性。同时,我们还通过检测不同条件下合成的HP-HSA期蛋白末端碱性氨基酸(主要包括赖氨酸及精氨酸)残基被HP修饰比率,比较HP-HSA的抗病毒活性与蛋白末端碱性氨基酸被修饰比率之间的相关性。
结果发现,HSA蛋白末端的碱性氨基酸残基大部分能被HP所修饰,其蛋白的化学结构发生了变化,从而将无活性的HSA转变成具有较强抗HIV-1活性的病毒抑制剂HP-HSA。HP-HSA中赖氨酸和精氨酸残基的被修饰的比率越高,其抗抗HIV-1X4型(HIV-1ⅢB)及R5型(HIV-1Bal)的病毒株的作用也相应的越强,说明蛋白末端碱性氨基酸被修饰的比率与HP-HSA抗病毒活性密切呈正相关,修饰蛋白末端的碱性氨基酸残基被相应酸酐修饰的比率与酸酐修饰蛋白的抗病毒活性密切相关。通过比较分析确定,HP-HSA化学修饰的最优合成条件为,酸酐HP终浓度60mM,pH值为9.0,在这一条件下,HP-HSA的蛋白末端赖氨酸被修饰的比率为98.17%,而精氨酸被修饰的比率为91.67%。而在这一条件下,合成的HP-HSA的抗HIV-1IIIB(X4型)和BaL(R5型)病毒的IC50值分别是0.851nM和1.454nM。
2. HP-HSA抗病毒活性研究
作为一个理想的防治性传播性疾病的抗病毒制剂,首先要保证其具有广谱性和有效性,即能够高效抑制绝大部分与性传播疾病有关的病原体的感染。与3HP-β-LG相类似,酸酐修饰蛋白HP-HSA也具有广谱高效的抗病毒活性,其能抑制多种HIV-1实验室病毒株包括HIV-1X4型和R5型,IC50在低纳摩尔水平。
另外,HP-HSA也能有效抑制HIV-2感染,意味着HP-HSA也能被用于HIV-2病毒广泛流行的西非等国家。同时HP-HSA还能抑制SIV的感染[4]。由于SIV病毒能够感染恒河猴,因此利用SIV病毒阴道攻击恒河猴的动物模型,可先期评价HP-HSA在动物体内的抗病毒活性。
目前大多数的抗病毒药物,尤其是临床上应用最为广泛的逆转录酶抑制剂临床应用的局限性最主要的是由于HIV病毒耐药株的产生。HIV病毒需寄生于宿主细胞内,利用宿主细胞的代谢系统生存、增殖,并保持极强的生命力。HIV病毒在宿主细胞内不断复制,但是其复制的错配率极高且极易产生高变异性,这也是目前理想抗HIV病毒药物研发缓慢的主要原因之一。本文研究发现,HP-HSA对几类常见的抗病毒药物包括核苷类逆转录酶抑制剂AZT,非核苷逆转录酶抑制剂A17及HIV进入抑制剂T20的耐药病毒株,均具有非常好的抑制作用,其IC50值均在低纳摩尔水平,与抗HIV-1实验室病毒分离株的抗性相当。这弥补了这些目前临床应用的主要的抗病毒药物的不足,从而使其具备了被进一步开发的前景。如果将这类蛋白类药物与抗病毒药物联合应用,可能也将成为抗病毒药物研究的一个新的方向。
如前所述,生殖器单纯疱疹病毒II型(Herpes Simplex Virus, HSV-2)是另一类导致性传播性疾病STD的主要病原体,约75%生殖器疱疹病例是由HSV-2引起的,它是引发生殖器疱疹和溃疡最主要的原因,是一个世界性的公共卫生问题。值得注意的是,其引发的生殖器疱疹据报道与HIV感染的关系极为密切,研究发现患有生殖器疱疹的病人更容易感染HIV,其HIV感染率可增加2-4倍。因此,有效控制HSV-2感染对减少HIV的流行具有直接的作用。如果抗病毒药物在具有抗HIV活性的同时,具有抑制HSV-2病毒的作用,则将大大增加该抗病毒药物的抗HIV活性,从而成为高效双功能的抗病毒制剂。目前,临床应用的大部分抗HIV药物均为窄谱抗病毒药物,其无或者对HSV-2的抑制作用不强。本文我们研究发现,酸酐修饰人血清白蛋白HP-HSA对HSV-2病毒有明显抑制作用。
3. HP-HSA抗病毒作用机制研究
HIV病毒进入CD+4靶细胞一般是在1h内完成的。如果一种药物为HIV进入抑制剂,其必须在病毒进入靶细胞前与病毒或靶细胞接触。因此,我们选用time-of-addition的分析方式,在病毒感染靶细胞的不同阶段加入酸酐修饰蛋白,以观察HP-HSA到底作用在HIV感染的哪个环节。实验结果发现,当HP-HSA在病毒感染2h后加入时,其抗HIV-lHIB和抗HIV-1BaL病毒活性完全消失,其作用与FDA批准的三种HIV进入抑制剂如T20,AMD3100(CXCR4辅助受体抑制剂)及Maraviric (CCR5辅助受体抑制剂)药物的失活时间相似,而对照药物逆转录酶抑制剂AZT,即使在病毒感染靶细胞8h后加入,仍然能保持较高的抗HIV-1活性。这些结果说明HP-HSA是一类特异性的HIV进入抑制剂,对HIV病毒复制的其他环节无明显作用。
由于阻断HIV病毒生命周期的第一个阶段,因此靶向HIV进入阶段的抗病毒药物均具有明显的优势。研究表明,游离病毒和已感染HIV的靶细胞都能感染新的靶细胞,而后者在HIV的感染中占主要地位。在HIV的感染过程中,首先是HIV包膜与靶细胞膜发生融合,HIV基因组进入靶细胞。HIV进入靶细胞是由病毒表面的包膜糖蛋白介导的。因此,阻止HIV病毒的进入,关键是阻止HIV Env诱导的细胞-细胞融合及病毒-细胞融合过程。通过细胞-细胞融合、病毒-细胞融合及细胞间的传播等实验结果分析可知,HP-HSA的确作用于病毒的进入阶段,是一类HIV进入／融合抑制剂。
HIV通过三个步骤进入宿主细胞：1)HIV颗粒通过其包膜上的糖蛋白gp120与靶细胞表面的CD4受体粘附；2)gp120发生构象改变以适应与靶细胞上的辅助受体CCR5或CXCR4结合；3)HIV跨膜蛋白亚基gp41介导病毒包膜与靶细胞膜发生融合。那么HP-HSA到底作用于HIV进入的哪个环节,则成了我们下一步关心的问题,因此我们对HP-HSA的作用靶点进行研究。
HIV进入靶细胞的第一步就是HIV病毒颗粒通过其包膜糖蛋白gp120与靶细胞表面的CD4受体粘附,因此,我们首先检测HP-HSA是否抑制gp120和可溶性CD4分子(sCD4)的体外结合。结果发现,HP-HSA能抑制来源于HIV-1HIB和HIV-1BaL的gp120与sCD4的结合,而未修饰HSA则无抑制作用。这些结果表明,HP-HSA可能是通过抑制病毒包膜糖蛋白gp120结合到靶细胞上的CD4受体而产生抑制作用的。ELISA和流式分析结果进一步表明,HP-HSA可通过与病毒包膜糖蛋白gp120和靶细胞上的CD4受体结合,阻止HIV-1的gp120与CD4两者的相互作用,从而产生抑制病毒与细胞膜融合的过程。HSA蛋白表面正电荷的精氨酸和赖氨酸残基被酸酐修饰之后,其表面聚集着大量的负电荷侧链,这些负电荷侧链能与gp120和CD4分子结合,从而产生抗病毒作用。
4. HP-HSA的局部用药安全性研究
理想的抗病毒药物除了具备上述价廉和高效等条件外,更为重要的一点即为其安全性。抗病毒药物由于可能每日被多次使用,且长期甚至终生服用,因此必须不会导致组织损伤,不会产生过敏反应,不会改变阴道内酸性环境,不会造成阴道内菌群失调,同时,应无全身性毒副反应。在体液中仍能保持较好的抗病毒活性。
阴道粘膜是多层鳞状上皮细胞,它们组成了防御外来病毒的一道物理屏障。因此,任何能破坏上皮组织完整性的因素,譬如物理损伤、性传播病原体的感染、局部溃疡及炎症、激素水平失衡、阴道正常菌群失调、以及不当使用阴道洗液等,都可能提高HIV的感染率。因此,药物对阴道上皮细胞的毒性是其能否用于临床的关键。我们首先评价了HP-HSA对3种T淋巴细胞和3种正常人阴道及子宫颈上皮细胞的体外毒性,结果显示,HP-HSA对测试的6种病毒作用T淋巴靶细胞及正常人阴道及子宫颈组织细胞的细胞毒性作用很低,其CC50的值远远大于其抑制HIV-1HIB病毒的IC50值,这意味着HP-HSA体外基本无毒。
阴道乳酸菌是内源性的杀微生物剂,它能通过产生乳酸和过氧化氢来发挥效能。流行病学研究表明产过氧化氢乳酸菌能有效降低HIV的感染率。其原因推测是间接的,产过氧化氢乳酸菌能降低细菌性阴道炎的发病率,而后者导致的阴道上皮组织损伤是HIV传染的一个主要影响因素。因此,我们评估了HP-HSA对17株正常人阴道乳酸杆菌生长的影响。药敏实验结果发现,较高浓度的HP-HSA对阴道正常乳酸杆菌的生长无明显影响,而阳性对照药物氨苄青霉素对阴道乳酸杆菌各株均有明显抑制作用,其MIC为0.313～1.25
前面研究证实,HP-HSA的作用靶点之一为T淋巴细胞表面的CD4受体,而CD4+T淋巴细胞是人免疫系统中极为重要的一类细胞,对人正常免疫功能起着至关重要的作用[8]。那么,HP-HSA作为杀微生物剂用于机体后,是否会对T淋巴细胞的正常的功能产生影响?首先,我们观察了HP-HSA是否对正常人PBMCs增殖的影响,同时观察了HP-HSA对PHA刺激的PBMCs增殖的反应,结果证明即使在浓度为100μM时,HP-HSA对正常和PHA刺激的PBMCs的生长增殖均无明显的影响。随后,我们评估了HP-HSA对正常和PHA刺激的PBMCs分泌细胞因子IFN-γ功能的影响,结果同样证明HP-HSA对正常和PHA刺激PBMCs的免疫功能无影响。这些结果初步证明,HP-HSA对CD4+T细胞的功能,尤其是处于体内循环状态的T细胞不会产生有害的影响。但是,我们也不能排除长期应用阻断CD4受体的杀微生物剂可能会抑制阴道粘膜中的CD4+T细胞的免疫功能,这也正是我们下一步准备进行的安全性实验,评估HP-HSA对家兔阴道粘膜免疫系统的影响。
性传播是目前HIV传播的主要途径,因此人体精液和宫颈阴道液对抗HIV药物活性影响的评估就显得至关重要了。一般来说,理想的抗病毒药物还应该在存在大量体液时,仍能保持较强的抗病毒活性。有文献报道,精液的存在会降低一些候选杀微生物剂的活性。本文结果可见,人体正常精液和阴道模拟液对HP-HSA的抗病毒活性并无显著影响。(五)小结
HP-HSA为水溶性蛋白,其无味,高浓度是呈淡黄色,制备简单、快速、可重现,且其体外毒性、免疫功能和稳定性评估证明其安全稳定,具有被开发成抗病毒药物的潜能。HP-HSA的优势：1)具广谱抗病毒活性,能对抗HIV-1,HIV-2, SIV和HSV的感染；2)能有效对抗各类药物的耐受株,包括核苷逆转录酶抑制剂耐药株、非核苷逆转录酶抑制剂耐药株及T20耐药株；3)HP-HSA为来源于人的大分子蛋白类药物,其生物半衰期长；4)HSA来源于人类,其抗原性较低,不易诱导不利的免疫反应。然而,HP-HSA作为蛋白类药物可能存在价格较高、给药方式较难选择等缺陷。将两类不同作用机制或不同作用靶点的新型抗病毒药物与临床应用的抗病毒药物联合应用,将有效避免各自的优缺点,取长补短,发挥各自的优势,从而产生协同作用效果。
Since the first cases of HIV/AIDS were reported in1981in the United States, AIDS is spreading worldwide rapidly. Theoretically, the best method to preventing HIV infection is to develop an HIV vaccine. However, there is still no effective HIV vaccine to be used in human beings. Currently, the major treatment for preventing HIV infection is the antiviral drugs. Until now,29antiviral drugs with different mechanism of actions have been approved by Food and Drug Administration (FDA). Most of them are belongs to two classes. One is reverse transcriptase inhibitor and the other is protease inhibitor. In spite of so many kinds of anti-HIV drugs used in patients, HIV can not be killed clearly. Those drugs can only suppress viral replication, reduce viral load and partly restore a patient's immune function and prolong the lives of patients. In2003, the first HIV entry inhibitor, Enfuweite (T-20), was approved by FDA, which is a peptide derived from HIV envelope protein gp41. HIV entry inhibitor can prevent the virus invasion at the first stage. However, it only specifically inhibits HIV-1infection but not inhibiting HIV-2infection. Otherwise, peptide drugs have expensive price, which also limit the clinical application. Another thorny issue is the high mutation of HIV virus. It is extremely easy to produce drug-resistant strains, which can reduce the antiviral activities of most of antiviral drugs. The highly active antiretroviral combination therapy (Highly Active Anti-Retroviral Treatment, HAART) is always combining with three or four kinds of antiviral drugs (typically containing a protease inhibitor). It has become a standard method of treating HIV infection, which is significantly reduced the mortality and morbidity of HIV-infected patients. HAART is also called as "cocktail therapy". A combination of different mechanisms of antiviral drugs may have the following advantages:(i) maximizing antiviral efficacy because of the potent synergistic activity against HIV-1laboratory-adapted both X4and R5strains,(ii) minimizing toxic effects and high cost due to combining dose reduction, and (iii) fighting against diverse anti-HIV-1drug mutations or delaying the emergence of HIV-1resistance by using agents with different mechanisms. Therefore, the devlepment of effective, safe and affordable new antiviral drugs to preventing HIV transmission is urgently needed.
Human serum albumin (HSA) is the most common protein in human plasma. It has a lot of important functions of physiology and pharmacology. Now HSA has become one of the most extensive target proteins in basic and clinical research, especially on appropriate chemical modification. Development a chemically modified product as an ideal therapeutic molecule has become one of the important means of research in life sciences. The non-glycosylated single polypeptide chain of HSA contains585amino acids and a relative molecular weight was66.5kD. It is mainly composed of18kinds of amino acid residues, in which contains59lysine and27arginine. Since the anhydride is a strongly acidic substance, the protein containing many alkaline amino acids can be modified by the anhydride. After chemical modification, an anhydride-modfied protein will be produced.
In1995, Neurath AR and Shibo Jiang in our group of New York Blood Center demonstrated that bovine β-lactoglobulin, a protein present in milk and whey, modified by3-hydroxyphthalic anhydride (3HP-β-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1and HSV-2). Nether non-modified B-lactoglobulin nor3-hydroxyphthalic anhydride had the anti-HIV activity. It means the chemical modification converted commonly available proteins into potent antiviral compounds.3HP-β-LG is highly stable in aqueous solution for long-term storage at room temperature and elevated temperatures. However, the outbreak of bovine spongiform encephalopathy (BSE) in Europe raised a safety concern with regard to developing bovine proteins for medical use, resulting in discontinuation of further development of3HP-β-LG as a microbicide.
HS A is the main protein in human blood, and it is composed of more than60%of the total proteins in the plasma. The structure of HSA is stable and it widely distributed in the human vascular and extravascular circulation. The half life of HSA is up to19days. Because of its human-derived protein, it is no obvious immunogenicity. Therefore, the anhydride modified HSA has the superiority to be developed an ideal antiviral drug in future. Therefore, we sought to replace bovine proteins with chemically modified HSA using the same method and the same conditions as3HP-β-LG Its antiviral activity and its possible clinical applications will be evaluated in the present study.
Optimization of the conditions for chemical modification of HP-HSA
HP-HSA was prepared according to the procedures previously described. For Optimization of the conditions for chemical modification of HP-HSA, we prepared HP-HSA under six different sets of conditions and evaluated the correlation between the anti-HIV-1activities (both X4and R5) and the percentage of lysine and arginine residues in HP-modified and-unmodified HSA to optimize the conditions for production of HP-HSA. Results showed that, with the increased concentration of HP (20,40and60mM, pH9.0) and the pH of the reaction system (7.0,8.0and9.0, concentration of HP is60mM), a higher percentage of lysine and arginine residues was modified, leading, in turn, to stronger anti-HIV-1activity on both IIIB (X4strain) and BaL (R5strain) viruses. Based on these results, we selected as the optimal condition (60mM of HP at pH9.0) for preparation of the HP-HSA. Under such condition, the EC50s of HP-HSA are about0.851and1.454nM on ⅢIB and BaL viruses, with98.17%and91.67%of the lysine and arginine residues modified by HP, respectively (Table1).
Determination of the antiviral activities of HP-HSA
It is desirable that a topical antiviral drug should be a broad-spectrum and effective antiviral agent. By ELISA, HP-HSA effectively inhibited infection by laboratory-adapted HIV-1strains, including X4and R5viruses, with IC50in the nM range.
HP-HSA is also effective in inhibiting HIV-2infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2is prominent. Our studies also showed that HP-HSA could potently inhibit infection by SIV. Results showed that HP-HSA strongly inhibited the infection by HIV-2ROD, and SIVmac25132H viruses, with low EC50s in the range of8.582to65.769nM Since SIV can be used for infection of rhesus macaques, HP-HSA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SIV infection through vaginal challenge..
Still, one of the most prominent problems facing future antiviral drugs is the development of resistant HIV-1strains against reverse transcriptase inhibitors. Our evidence demonstrated that HP-HSA retained high activities against different resistant HIV-1variants, including NRTI-resistant Zidovudine-R, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant A17-R, and HIV-1entry inhibitor-resistant NL4-3(36G)V38A and NL4-3(36G)V38E/N42S (Table1). Those results indicate that HP-HSA is capable of preventing the sexual transmission of HIV-1strains that are resistant to the currently used antiretroviral therapeutics.
As we described above, herpes simplex virus-2(HSV-2) produces most genital herpes, which is primarily associated with HIV transmission. Clinical data indicate that patients with prevalent HSV-2infection have a two to four-fold higher risk of HIV acquisition than HSV-2uninfected population. Therefore, we investigated the efficacy of HP-HSA against HSV-2in vitro in this study. Results showed that HP-HSA also has very low inhibitory activity on HSV-2and the ECsos value was34.514nM. The negative control protein, unmodified HSA even at concentrations up to10.000uM, had no inhibitory activity against any of the above viruses (data not shown). Cell viability analysis results showed that HP-HSA at the concentration of the effective concentration for100%inhibition (EC100) and lower than EC100had no cytotoxicity to virus target cells (Vero cells).
Evaluation the possible mechanism of HP-HSA on HIV-1infection.
As described before, HP-HSA exhibited significantly decreased inhibitory activity when it was added to target cells0.5-2h post-HIV-1infection by time of addition assay.6While such results indicated that HP-HSA was an HIV-1entry/fusion inhibitor, the potential targets of HP-HSA remained unknown. Therefore, to systematically search for these targets, we examined the effect of HP-HSA on the early steps of the HIV-1replication cycle, i.e., HIV-1fusion/entry, using cell-cell fusion and virus-cell fusion assays.
HIV-1Env--mediated cell-cell fusion was detected using fusion of noninfectious CHO-WT cells with MT-2cells or infectious HIV-1ⅢB-infected H9cells (H9/HIV-1Ⅲb cells) with MT-2cells, respectively. In noninfectious analysis system, the giant syncytia were observed by an inverted microscope. The size of syncytium is usually twice or more times than the size of normal cells. In infectious analysis system, the fluorescence diffuses were observed by an inverted fluorescence microscope. The numbers of the formation of giant syncytia and fluorescence diffuses in cell-cell fusion assay were counted by choosing four fields randomly under an inverted microscope. HP-HSA could significantly decrease the formation of giant syncytia and fluorescence diffuses in a dose-dependent manner. The percentage of inhibition of cell-cell fusion of HP-HSA and HSA were calculated according to the formula described above. Results showed that HP-HSA inhibited both kinds of HIV-1Env-mediated cell-cell fusion, with EC50about27.82nM in a noninfectious system and7.45nM in an infectious system, respectively. In the above-described assays, unmodified HSA demonstrated no ability to inhibit cell-cell fusion.
A single-round entry assay was chosen to detect the inhibition of HP-HSA on direct virus-cell fusion by using pseudotyped viruses expressing HIV-1HXB2(X4strain) and SF162(R5strain) Env. Similar results were observed in this assay. HP-HSA inhibited infection by both X4and R5Env-pseudotyped viruses, with EC50about1.51and1.54nM, respectively. Vesicular stomatitis virus-G (VSV-G) pseudovirus was produced by cotransfecting VSV envelope G-protein plasmid and pNL4-3E-R-Luc plasmid. Therefore, VSV-G pseudovirus could be used to evaluate the specific effects of HP-HSA on HIV-1envelope proteins. Results showed that VSV-G pseudovirus infection could not be inhibited by HP-HSA, even at the concentration as high as1.25Ｌ.M
Cell-to-cell transmission is the major route for the spread of HIV-1. Here, we detected the inhibition of HP-HSA on the transmission of cell-associated HIV-1virions from PBMCs to CEMxl745.25M7cells. HP-HSA blocked the cell-cell HIV-lBaL transmission between those two kinds of virus target cells, which suggests that it could prevent the transmission of cell-associated HIV-BaL isolates. The EC50of cell-cell transmission was31.01nM. Unmodified HSA had no inhibitory activity on cell-cell transmission.
All of these results indicate that HP-HSA can inhibit HIV-1infection by blocking the virus entry step. HIV-1entry into a CD4+T cell is mediated by viral envelopes, which comprise the surface subunit gpl20and transmembrane subunit gp41. Similar to HP-OVA and ML-OVA, one of the probable mechanisms of HP-HSA might be interference with the viral envelope glycoprotein or the receptor on the target cell membrane.
Evaluation the safty of HP-HSA
general, HIV transmission during sexual intercourse should be occurred in presence of normal human body fluids, such as seminal and vaginal secretions. The antiviral activities of an ideal antiviral drug should be retained in the presence of those human body fluids. Here, we determined the potential effect of SP and VFS on the anti-HIV activity of HP-HSA. Results showed that neither SP nor VFS had significant effect on the inhibitory activity of HP-HSA against infection by both HIV-1X4(ⅢB) and R5(BaL) strains. The EC50values of HP-HSA on HIV-1ⅢB strains in the presence of PBS, SP or VFS were1.605nM,1.532nM or1.408nM, respectively. Moreover, the EC50values of HP-HSA for inhibiting HIV-1BaL infection in the presence of SP and VFS were4.997nM and7.564nM, respectively, whereas that of PBS control is6.477nM.
Lactobacilli are the predominant organisms in the healthy vagina of women, which maintain a normal vaginal microbiota and form a natural physical barrier against other bacteria or viruses. Here, we chose17strains of normal human vaginal lactobacilli and evaluated the effects of HP-HSA on the proliferation of those vaginal lactobacilli. Results showed that HP-HSA had not produced significant inhibitory effect on17strains of vaginal lactobacilli (MIO15.000μM). The MICs of positive control Ampicillin on different strains were from0.841μM to6.730μM.
Normal CD4+T lymphocytes in human beings are essential in the immune system, such as helping antigen presenting cell expresses an antigen on MHC class II through a combination of cell-to-cell interactions or through cytokines.32As we described above, since HP-HSA can bind to cellular CD4receptor, a concern is raised about the function of CD4+T lymphocytes in the presence of HP-HSA.33In our previous studies, we confirmed that HP-HSA had no significant effects on the proliferation of T lymphocytes and no obvious effects on the production of IFN-y in either human PBMCs or PHA-stimulated PBMCs. One advantage of HP-HSA is its origin from human sera, which may reduce any deleterious effects on the function of CD4+T cells, especially for those circulating in the blood stream. Nonetheless, the long-term use of CD4blockers could still suppress the function of CD4+T cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-HSA on the mucosal immune system is warranted.
Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-HSA is broad-spectrum HIV entry/fusion inhibitors through blocking viral entry. By its broad antiviral potency, resistance to trypsin hydrolysis, easy preparation, low production costs, wide availability and absence of carcinogenic phthalic group, HP-HSA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-HSA may be used in combination with an NNRTI-based microbicide for preventing sexual transmission of HIV because the combination may have synergistic antiviral activity against a broad spectrum of HIV-1strains and reduce the potential toxic effects.These findings might be helpful to looking for a novel strategy for treatment of HIV/AIDS and might provide a rational basis for testing of microbicide combinations in vivo.
引文
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[26]Li M, Li L, Jiang S, Zhang J. A non-infectious assay for detecting HIV-1 Env-induced cell-cell fusion (in Chinese). J Jinan Univ (Med Sci) 2008; 28(6):576-580.
[27]Jiang S, Debnath AK. Development of HIV entry inhibitors taregeted to the coiled coil regions of gp41. Biochem Biophys Res Commun 2000; 269(3):641-646.
[28]Geuenich S, Goffmet C, Venzke S, Nolkemper S, Baumann I, Plinkert P, et al. Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density. Retrovirology 2008; 5:27.
[29]Neurath AR, Strick N, Li YY, Debnath AK. Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120. BMC Infect Dis 2001; 1(1):17.
[30]VanCott TC, Mascola JR, Kaminski RW, Kalyanaraman V, Hallberg PL, Burnett PR, et al. Antibodies with specificity to native gpl20 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160. J Virol 1997; 71(6):4319-4330.
[31]Zhou J, Swiderski P, Li H, Zhang J, Neff CP, Akkina R, et al. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells. Nucleic Acids Res 2009; 37(9):3094-3109.
[32]Yu H, Tudor D, Alfsen A, Labrosse B, Clavel F, Bomsel M. Peptide P5 (residues 628-683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection. Retrovirology 2008; 5:93.
[33]Lu M, Kim PS. A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein. J Biomol Struct Dyn 1997; 15(3):465-471.
[34]Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22:27-55.
[35]Boscolo P, del SA, Sabbioni E, Di GM, Di GL, Reale M, et al. Effects of resveratrol on lymphocyte proliferation and cytokine release. Ann Clin Lab Sci 2003;33(2):226-231.
[36]Lewis E, Rogachev B, Shaked G, Douvdevani A. The in vitro effects of ketamine at large concentrations can be attributed to a nonspecific cytostatic effect. Anesth Analg 2001; 92(4):927-929.
[37]Fernandez-Romero JA, Thorn M, Turville SG, Titchen K, Sudol K, Li J, et al. Carrageenan/MIV-150 (PC-815), a combination microbicide. Sex Transm Dis 2007; 34(1):9-14.
[38]Fletcher PS, Harman SJ, Boothe AR, Doncel GF, Shattock RJ. Preclinical evaluation of lime juice as a topical microbicide candidate. Retrovirology 2008; 5:3.
[39]Xiong W, Zhao Y, Prall A, Greiner TC, Baxter BT. Key roles of CD4+ T cells and IFN-gamma in the development of abdominal aortic aneurysms in a murine model. J Immunol 2004; 172(4):2607-2612.
[40]UNAIDS.2008 Report on the global AIDS epidemic. http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/2008/2008_Global_report.asp; 2008.
[41]Cutler B, Justman J. Vaginal microbicides and the prevention of HIV transmission. Lancet Infect Dis 2008; 8(11):685-697.
[42]Van DL, Govinden R, Mirembe FM, Guedou F, Solomon S, Becker ML, et al. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med2008; 359(5):463-472.
[43]Jakubowski A, Maksimovich N, Olszanecki R, et.al. S-nitroso human serum albumin given after LPS challenge reduces acute lung injury and prolongs survival in a rat model of endotoxemia [J]. N-S Arch Pharmacol,2009, 379(3):281-290.
[44]KATAYAMA N, NAKAJOU K, KOMORI H, et al. Design and evaluation of S-nitrosylated human serum albumin as a novel anticancer drug [J]. J Pharmacol Exp Ther,2008,325(1):69-76.
[45]Carter D.C. and He Xiao-Min et al, Science,1989,244:1195.
[46]He Xiao-Min and Carter D.C., Nature,1992,358(16):209.
[47]Kinghorn GR. Epidemiology of genital herpes. J Int Med Res,1994,22(Suppl 1):14A-23A.
[48]马瑜。单纯疱疹病毒侵入相关糖蛋白的研究进展。国际病毒学杂志[J],2006,13(3)：94-96。
[49]雷漾。产H2O2的乳酸杆菌在性传播疾病中的作用。中国艾滋病性病[J],2007,13(2)：193-94。
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