酸酐修饰卵清蛋白作为候选杀微生物剂预防HIV性传播研究
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摘要
目的：尽管预防和治疗HIV病毒感染的研究手段不断地发展和完善,但全球HIV/AIDS感染人群的比例仍在持续增长,艾滋病也正成为威胁人类健康的第一大病因的传染性疾病。目前,全球每天AIDS新增感染病例为7400人,每天AIDS导致死亡人数为5500人。HIV的传播途径主要有三种：血液传播、母婴传播和性传播。最新研究报道,无保护的性交已经成为当前感染HIV最为主要的途径,尤其是对于许多发展中国家的女性。这些国家的女性往往地位较为低下,在性交过程中没有能力要求其性伴侣采用保护措施。具统计,目前新增HIV感染的病例中,女性占较大比例。因此,发展女性可控制的预防性传播性疾病的杀微生物剂(Microbicides),已是迫在眉睫。
杀微生物剂是一类含有抗HIV成分的凝胶、乳脂、栓剂、药膜或海绵,在性交前置入阴道或肛门内,可预防艾滋病病毒和其它性传播疾病病毒的传播。杀微生物剂可通过直接灭活HIV、阻止HIV粘附及HIV侵入阴道或直肠粘膜内的靶细胞、抑制HIV在靶细胞内的复制等作用机理,保护健康人群在性行为时免受HIV感染。
理想的杀微生物剂应具备以下条件：广谱、安全、高效、在人体液中仍能保持较高抗病毒活性,对游离的病毒颗粒和细胞携带的病毒都应有抑制作用,对阴道、子宫颈或直肠粘膜上皮组织无损伤作用,对阴道共生菌群尤其是乳酸菌无抑制作用,具有耐酸、耐热、无色无味、价格低廉、易于使用等优点。基于这些特点,本文旨在寻找一类新型具特异性抑制病毒作用的候选杀微生物剂,用以预防和治疗HIV的性传播。
1995年,美国纽约血液中心的Neurath AR和姜世勃等人研究发现3-羟基-邻苯二甲酸酐(3-hydroxyphthalic anhydride,3-HP)修饰的p-乳球蛋白(p-lactoglobulin, P-LG),即3-HP-p-LG,可特异性抑制HIV的感染。而未经修饰的β-乳球蛋白及3-HP本身均无抑制HIV的活性,说明酸酐化学修饰将无活性的蛋白质转化成了具有抗HIV活性的病毒抑制剂。进一步研究还发现,3HP-β-LG具有极为广谱抗病毒活性,可有效对抗各种HIV-1实验室病毒株、HIV-1临床分离株、HIV-2和猿免疫缺陷病毒(simian immunodeficiency viruses, SIV)的感染。值得关注的是,3HP-β-LG还对其它性传播感染性病毒,如单纯疱疹病毒(HSV),沙眼衣原体(Chlamydia trachomatis)等,也具有较强的抑制活性。由于3HP-β-LG具有广谱、价廉、高效、稳定等特点,使其具备了被开发成杀微生物剂的条件。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-p-LG的研发受到了极大的影响,将其作为杀微生物剂研发的试验研究被迫终止。
本文旨在寻找一类新的非牛来源的蛋白来替代β-乳球蛋白,采用与3HP-B-LG相似的酸酐修饰方法,合成出新的酸酐修饰蛋白,通过对其抗病毒活性的分析,寻找出最具开发潜力的预防HIV性传播的候选杀微生物剂应用于临床。基于这一基本思路,本文选用了5种价格低廉、安全无毒的非牛奶来源的蛋白,包括：卵清蛋白OVA、兔血清白蛋白RSA、猪血清白蛋白PSA、冷水鱼皮明胶G-FS和猪皮明胶G-PS,作为被修饰对象,制备出一系列酸酐修饰蛋白,以期筛选出一类具有高效、低毒、价廉和稳定的候选杀微生物剂进行研发。
方法与结果：首先,选用3-羟基-邻苯二甲酸酐HP分别修饰上述5种动物蛋白,经化学合成方法合成5种不同的酸酐修饰蛋白(HP-OVA、HP-RSA、HP-PSA、HP-G-FS及HP-G-PS)。通过ELISA方法检测这5种酸酐修饰蛋白体外抗HIV-1 X4型(HIV-1ⅢB)及R5型(HIV-1Bal)病毒的活性。结果发现5种酸酐修饰蛋白具有不同程度的抗HIV-1病毒活性,其中抗病毒活性较高的为HP-OVA、HP-RSA和HP-PSA。比较分析这些酸酐修饰蛋白的抗病毒活性与被修饰蛋白的空间结构关系发现,OVA、RSA和PSA与β-乳球蛋白的结构相似,其空间结构均为球蛋白,而抗病毒活性较弱的被修饰蛋白G-FS和G-PS则是属于片层结缔状结构的明胶蛋白。由此可知,酸酐修饰的球蛋白的空间结构可能有助于其抗病毒活性的提高。3种动物蛋白(OVA、RSA和PSA)经HP酸酐修饰后虽都能转变成HIV抑制剂,我们选用卵清蛋白OVA作为后续研究蛋白,其主要原因是,OVA为鸡蛋清中的主要蛋白,占蛋清中总蛋白含量的60-65%,其来源广泛、价格低廉,极有可能被开发成具有自主知识产权、便宜、高效、应用简便的阴道用杀微生物剂,用于预防我国HIV的性传播。相比之下,其它两类蛋白均来源于血液,价格相对较高,且用药后具有潜在的感染其他病原体的危险性。
为了比较不同酸酐修饰OVA抗病毒活性的变化,本文选用3种酸酐：饱和酸酐(琥珀酸酐SU),不饱和酸酐(马来酸酐ML)及芳香族酸酐(3-羟基邻苯二甲酸酐HP)分别修饰OVA,得到3种酸酐修饰的卵清蛋白SU-OVA、ML-OVA和HP-OVA。通过比较这3种修饰蛋白的抗HIV-1活性,发现芳香族酸酐HP修饰蛋白后的产物HP-OVA的抗病毒活性最强,其次为不饱和酸酐修饰产物ML-OVA,最差为饱和酸酐修饰产物SU-OVA。由此可初步判定,酸酐中的苯环及不饱和烯键能明显提高酸酐修饰蛋白的抗病毒活性。综合上述结果,我们选用芳香族酸酐HP修饰的HP-OVA作为后续实验研究对象。
为优化HP-OVA的制备条件,我们用不同浓度的HP和不同的pH值反应体系,合成了的一系列不同的HP-OVA,并评价这些HP-OVA的抗病毒活性与蛋白末端碱性氨基酸残基被HP修饰比率的关系。结果发现,OVA蛋白末端的碱性氨基酸残基大部分能被HP所修饰,从而将无活性的OVA转变成具有较强抗HIV-1活性的病毒抑制剂HP-OVA。HP-OVA中赖氨酸和精氨酸残基的被修饰的比率越高,其抗HIV-1 X4和R5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-OVA抗病毒活性密切呈正相关。通过比较分析确定,HP-OVA化学修饰的最优合成条件为,酸酐HP终浓度40mM, pH值为8.5,在这一条件下,HP-OVA的蛋白末端赖氨酸被修饰的比率为99.86%,而精氨酸被修饰的比率为89.26%。
作为候选杀微生物剂,首先要具有广谱的抗病毒活性。经ELISA检测,HP-OVA具有极为广谱的抗病毒活性,能高效抑制多种HIV-1实验室病毒株包括HIV-1 X4型和R5型病毒感染,其IC5o均在低纳摩尔水平。同时,HP-OVA能高效抑制FDA批准的抗HIV药物,HIV进入抑制剂T20及逆转录酶抑制剂AZT耐受病毒株的感染,这为我们提供了一个新的治疗思路,即将HP-OVA与这些抗病毒药物合用,可能会产生取长补短的协同作用,这也是我们后续实验内容之一。
同时,HP-OVA也能高效地抑制各种HIV-1临床分离株的感染,包括A亚型-G亚型及O亚型(R5型或X4R5型),其IC50从0.011μM到0.578μM。特别是HP-OVA对HIV-1临床分离的A/E亚型病毒株感染具有很高的抑制活性,其IC5o仅为10 nM,而A/E亚型病毒株是性传播性疾病最为主要的传播病毒之
该病毒株在泰国较为流行,HP-OVA对该病毒株具有较好的抑制作用,预示着其在我国预防HIV性传播方面将会有较大的市场和应用前景。
另外,HP-OVA能有效抑制HIV-2感染,意味着HP-OVA也能被用在HIV-2病毒广泛流行的西非各国。与3HP-β-LG相似,HP-OVA也能对抗其他性传播性病原体HSV-2的感染。HP-OVA还能抑制SHIV和SIV的感染,由于SHIV和SIV病毒能够感染恒河猴,因此利用SHIV或SIV病毒阴道攻击恒河猴的动物模型,可先期评价HP-OVA在动物体内的抗病毒活性,为临床试验研究奠定了坚实的基础。
与目前临床试验失败的阴离子多聚体类候选杀微生物剂相比,HP-OVA的抗HIV-1病毒的活性,尤其是对HIV-1R5亚型病毒株的抑制作用明显增强,这极大地弥补了这些候选杀微生物剂的不足,从而使其具备被进一步开发的广阔前景。但HP-OVA为何能产生抗病毒活性,其作用机制如何,则是我们更为关注的问题。
通过time-of-addition、细胞-细胞融合、病毒-细胞融合及病毒细胞-细胞间的传播等实验结果分析可知,HP-OVA作用于HIV病毒的进入阶段,是一类HIV进入／融合抑制剂,其对HIV病毒感染靶细胞后期的复制过程无明显抑制作用。采用ELISA、SPR和流式细胞仪FCM分析还发现,HP-OVA通过与HIV包膜糖蛋白或病毒作用靶细胞上的CD4分子结合,干扰包膜糖蛋白与CD4分子的结合,从而抑制病毒与靶细胞膜的融合,阻止HIV的进入。通过ELISA和FN-PAGE方法,我们还发现较高浓度的HP-OVA对HIV跨膜糖蛋白gp41的六螺旋核心结构的形成也具有一定的抑制作用,从而抑制gp41介导的膜融合过程。这些结果说明,HP-OVA可能是一类多靶点的HIV进入抑制剂。
作为理想的杀微生物剂,除了高效之外,另一个必要条件是对人体安全无毒。体外安全性实验表明,HP-OVA对3种HIV病毒作用的T淋巴靶细胞及3种人正常阴道及子宫颈组织上皮细胞的细胞毒性均很低,其CC50的值远远大于其抑制HIV-1ⅢB病毒的IC50值,其选择系数(SI=CC50/IC50)范围为253到13066,说明HP-OVA体外对病毒靶细胞和组织上皮细胞基本无毒。阴道乳酸菌是一类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。作为外源性的杀微生物剂,不应该对阴道正常菌群的生长产生影响。因此,我们评价了HP-OVA对正常阴道乳酸菌生长是否有影响,结果发现22.4μM HP-OVA对17株正常人阴道乳酸杆菌的生长无明显抑制作用。
前述结果表明,HP-OVA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到至关重要的作用,HP-OVA用药是否会对人T淋巴细胞的功能产生影响呢?为此,我们首选观察了HP-OVA是否对正常人PBMCs及PHA刺激的PBMCs的增殖产生影响,结果证明,即使HP-OVA在浓度为100μM时,其对正常和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-OVA对正常和PHA刺激的PBMCs分泌细胞因子IFN-y功能的影响,结果同样证明HP-OVA对PBMCs的免疫功能无影响。这些结果初步表明,HP-OVA对CD4+T细胞的正常增殖和免疫功能,尤其是处于体内循环状态的T细胞不会产生有害的影响。
蛋白和多肽类药物,如T20,其主要的缺点之一为体内半衰期较短,极易被体内的蛋白酶水解。胰蛋白酶就是人体内重要的代谢蛋白酶之一,其在阴道菌群中也大量存在。胰蛋白酶裂解蛋白的主要位点是蛋白或多肽氨基酸末端的精氨酸或赖氨酸。由前述结果可知,OVA蛋白末端的碱性氨基酸,包括赖氨酸和精氨酸,绝大部分被HP酸酐所修饰,OVA蛋白末端氨基酸残基中胰蛋白酶的裂解位点有可能因酸酐修饰而发生改变。因此,我们检测了HP-OVA对胰蛋白酶的稳定性。结果表明,HP-OVA在胰蛋白酶的存在时,其抗病毒活性基本稳定,SDS-PAGE显示,蛋白结构也没有被酶所水解,而是保持相对稳定的分子量。
目前性传播已经成为HIV传播的主要途径,因此评估性传播中广泛存在的人体精液和宫颈阴道液对HP-OVA抗病毒活性的影响就显得至关重要了。本文结果可见,人体正常精液及阴道模拟液对HP-OVA的抗病毒活性并无显著影响。作为杀微生物剂制剂,样品的保存条件也不容忽视。我们将HP-OVA溶液保存于不同温度下,结果发现,不同温度对样品抗病毒活性无明显影响。
从2000年至2009年,几个候选杀微生物剂大规模临床Ⅲ期试验的相继失败,包括：Savvy (C31G), Cellulose sulfate (CS),0.5% PRO 2000凝胶及Carraguard等,使杀微生物剂研发前景不容乐观。而这些候选杀微生物剂临床试验失败的主原因之一被认为是由于它们对性传播过程中最为普遍的一类病毒株HIV-1 R5型病毒的抑制活性相对较低。为提高杀微生物剂的有效性,近年来研究的重点转向具有特异性、作用机制明确的第二代杀微生物剂。目前临床上研发的第二代候选杀微生物剂主要为逆转录酶抑制剂,如TMC-120, UC781和Tenofovir,但它们也有相应的缺陷,如极易诱发产生病毒耐药株等。HP-OVA具备广谱、高效、价格低廉、易于制备及安全稳定等特点,且对大部分药物产生的耐药株有较好的抑制活性。HP-OVA为多靶点的HIV进入抑制剂,故其临床应用可能不易产生耐药株。因此,本文选用一系列可能被发展成为候选杀微生物剂的抗病毒药物,包括HIV进入抑制剂和逆转录酶抑制剂,与HP-OVA合用,观察HP-OVA与各药单用及合用后的协同作用效果。结果发现,HP-OVA与各药合用时,均能产生不同程度的协同作用效果。HP-OVA与第二代候选杀微生物剂合用具有以下优点：1)增强抗病毒活性,扩大抗病毒谱；2)降低毒副作用,减少用药剂量；3)多靶点用药,延缓病毒耐药株的产生；4)降低药物成本,优化保存条件。
结论：酸酐修饰卵清蛋白HP-OVA具有高效广谱的抗病毒活性,能有效对抗各种HIV-1实验室病毒株和临床分离株的感染,同时还具有抑制HIV-2、SHIV、SIV及HSV-2的作用；机制研究表明,HP-OVA通过与HIV包膜糖蛋白或病毒作用靶细胞上的CD4分子结合,干扰包膜糖蛋白与CD4分子的结合,从而抑制病毒与靶细胞膜的融合,阻止HIV的进入；体外安全性及稳定性试验证明,HP-OVA对病毒作用靶细胞和阴道正常细胞安全无毒,其对阴道正常菌群的分泌及CD4+T淋巴细胞的增殖和免疫功能均无明显影响,胰蛋白酶及人体体液对其活性影响不大；其蛋白来源广泛、价格低廉,具备发展成理想的杀微生物剂的基本条件,可作为预防HIV性传播的杀微生物剂进行研发；HP-OVA能和具有开发成杀微生物剂潜能的抗病毒药物合用,具有提高疗效,减少药物毒副作用的协同作用效果,这为寻找新的预防HIV性传播治疗策略提供了较好的理论基础,也为杀微生物剂的临床开发提供新的思路。
Objection:Despite extraordinary advances in the development of prevention and therapeutic strategies against human immunodeficiency virus (HIV) infection, HIV/AIDS continues to spread at an alarming rate worldwide. There are approximately 7,400 new infections and over 5,500 new deaths resulting from AIDS each day. There are three main transmission routes for HIV, including by blood, mother to child and unprotected sex. Unprotected sex is the primary route for humans now, especially for females in many developing countries, to acquire HIV/AIDS. Therefore, development of female-controlled topical microbicides is urgently needed.
Micribicides are gels, creams, foams, or suppositories, which can be applied in vagina or rectum before sex to prevent sexual transmission of HIV. These products are designed to protecting HIV infection by killing HIV, inhibiting HIV entry target cells or HIV replication in the cells.
An ideal microbicide should be broad antiviral activity, safe, highly effective, affordable, stable in human fluids, and easy to use. It should be safe to human tissues and to vigina bacteria. Microbicides should also be cheap to be used in many developing countries. Based on these characters, our group is searching for some new anti-HIV microbicide candidates for preventing HIV sexually transmission.
In 1995, our group of New York Blood Center demonstrated that bovine B-lactoglobulin, a protein present in milk and whey, modified by 3-hydroxyphthalic anhydride (3HP-B-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1 and HSV-2). Nether non-modified B-lactoglobulin nor 3-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 of 3HP-B-LG as a microbicide.
Therefore, in the present study, we sought to replace bovine proteins with chemically modified animal proteins of non-bovine origin as new anti-HIV microbicide candidates. All of the non-bovine animal proteins including chicken ovalbumin (OVA), rabbit serum albumin (RSA), porcine serum albumin (PSA), gelatin from cold water fish skin (G-FS), gelatin from porcine skin (G-PS) were modified by 3-hydroxyphthalic anhydride (HP), using the same method and the same conditions as 3HP-B-LG.
Methods and Results:After extensive screening, we found that several HP-modified non-bovine-origin proteins exhibited inhibitory activity (HP-OVA, HP-RSA, HP-PSA, HP-G-FS, HP-G-PS) against infection by HIV-1 X4 (HIV-1ⅢB) and R5 (HIV-1BaL) viruses. By analyzing the structure of the proteins found to possess antiviral activity, OVA, RSA and PSA were found to have representative globulins identical to bovineβ-lactoglobulin. By contrast, the gelatins used in this study are derived from collagens, which had different structure and conformation. The absence of anti-HIV activities of these modified proteins indicated that HIV blocking abilities might not be solely dependent on the modified lysine or arginine but also on the protein conformation. Thus, the presence of specific globular structures might play an important role in the anti-HIV activity of OVA, RSA and PSA. y evaluating the anti-HIV activities of these modifications and the characteristics of proteins used in the reaction, we found that HP-modified chicken ovalbumin (HP-OVA) was the most promising anti-HIV inhibitor among these modified proteins. Chicken ovalbumin (OVA) is the main protein in egg white, making up 60-65% of the total protein. Since OVA is one of the most abundant proteins consumed by people worldwide and is a generally recognized as a safe (GRAS) protein, HP-modified OVA has great potential for further development as an effective, safe and affordable microbicide.
To search for alternate anhydrides as chemical modifiers of OVA, we selected two other anhydrides, succinic anhydride (SU) and maleic anhydride (ML), for the chemical modification. We compared the efficiency of three different anhydrides, including ML, SU, as well as HP, for the chemical modification of OVA. All three anhydrides (SU, ML and HP) were sufficiently potent to convert OVA into an effective anti-HIV agent. By compared the anti-HIV-1 activity of those three anhydride OVAs, HP-OVA and ML-OVA demonstrated more efficacy than SU-OVA in blocking HIV-1 infection, especially the sexually transmitted R5 virus. It is the chemical structure of anhydrates that accounts for the effect of different anhydride OVA modifications on HIV inhibitory activities. Specifically, the only difference between maleic and succinic anhydride was the double bond between C3 and C4 in maleic anhydride, which led to the stronger inhibition abilities of ML-OVA over those of SU-OVA on HIV infection.3-hydroxyphthalic anhydride has a hydrophobic aromatic group, leading to the most potent anti-HIV activity. These findings suggest that the aromatic and unsaturated structure in anhydrides might contribute to the difference in antiviral activities of these modified OVAs.
For confirming the optimal condition, we prepared a series of HP-OVA by changing the different HP concentration and different pH values. Here we found that the percentage of the HP-modified and unmodified lysine and arginine residues in OVA was dependent on the concentration of HP and pH of the reaction system and was correlated with the anti-HIV-1 activity of HP-OVA. These results suggested that the modified amino acid residues play an important role in mediating the antiviral activity. We selected 40 mM concentration of HP and pH 8.5 as the optimal condition for preparation of the HP-OVA for the subsequent studies. Under such condition, HP-OVA had 99.86% and 89.26% of the lysine and arginine residues modified by HP, respectively.
By ELISA, HP-OVA effectively inhibited infection by laboratory-adapted HIV-1 strains, including X4 and R5 viruses, with IC50 in the nM range. HP-OVA is highly effective in inhibiting the infection of the primary R5 viruses with distinct genotypes and phenotypes, with IC50 from 0.011μM to 0.578μM. HP-OVA is more effective against the predominant HIV-1 subtypes A, B, and C. Particularly, HP-OVA is highly potent against the primary HIV-1 isolated in Thailand,92TH009 (subtype A/E, R5) with IC50 about 10 nM, while it was reported that the HIV-1 subtype A/E R5 virus was preferentially sexually transmitted. Those results suggest that HP-OVA have a great potency to be used as a microbicide candidate in the world, especially in our country.
HP-OVA is also effective in inhibiting HIV-2 infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2 is prominent. Our studies also showed that HP-OVA could potently inhibit infection by SHIV and SIV. Since both SHIV and SIV can be used for infection of rhesus macaques, HP-OVA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SHIV or SIV infection through vaginal challenge.
Compared with several negatively charged polymeric microbicide candidates failed in clinical trials, most of these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. However, this is not the case for HP-OVA since it is almost equally effective against both X4 and R5 viruses. Next, we try to find the mechanism of HP-OVA for inhibiting the HIV infection.
By using time-of-addition, cell-cell fusion, and cell-to-cell transmission assays, we demonstrated that HP-OVA is an HIV entry/fusion inhibitor since it exhibited significantly decreased inhibitory activity when it was added after HIV-1 infection and it showed potent inhibitory activity on cell-to-cell fusion mediated by X4 virus and cell-to-cell transmission of R5 virus. Using ELISA, SPR and FCM assay, HP-OVA could block the binding of the HIV-1 Env surface subunit gp120 (from both X4 and R5 viruses) or an anti-CD4 antibody to sCD4, the primary receptor for HIV, resulting in inhibition of interaction between gp120 and CD4. Furthermore, we demonstrated that HP-OVA could block the formation of the fusion-active gp41 six-helix bundle. These results suggest that HP-OVA inhibit HIV-1 entry/fusion through multiple mechanisms of action by interacting with both gp120 and gp41 as well as CD4 via the negatively charged residues of HP-OVA.
The failure of microbicide candidates clinical trials warned us that the safety evaluation of a microbicide candidate should be carried out as early as possible. Here we first assessed the potential cytotoxicity of HP-OVA on three well-characterized cell lines derived from the human vaginal and cervical epithelium and three human T immune cell lines as well as PBMCs, which were used for evaluation of the in vitro anti-HIV-1 activity of HP-OVA. The results showed that HP-OVA had low cytotoxicity to all tested cells. Its selectivity index (SI=CC50/IC50) ranged from 253 to 13,066, indicating that HP-OVA appears to be safe in vitro. More extensive animal studies to evaluate its in vivo toxicity will be carried out in the future. Vaginal lactic acid bacteria are not be affected by HP-OVA, even in the high concentration in 22.4μM.
Since HP-OVA can bind to sCD4 while the CD4 molecule on the T lymphocytes plays an important role in T cell activation, one of the concerns is whether HP-OVA affects the function of CD4+ T cells or induces T cell anergy, a status of the lymphocyte that is functionally inactivated following an antigen stimulation. Our studies demonstrated that proliferation of T lymphocytes in human PBMCs stimulated with PHA was not significantly affected by HP-OVA at the concentration as high as 100μM. Further studies showed that HP-OVA had no significant effect on the productions of IFN-y by PBMCs with or without PHA stimulation. Those results suggest that HP-OVA may not have deleterious effects on the function of CD4+ T cells, especially for those circulating in the human body. But we cannot exclude the possibility that long-term use of CD4 blockers topically may suppress the function of CD4+ immune cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-OVA on the mucosal immune system is warranted.
The disadvantage of protein/peptide drugs, such as T20, is their short half life resulting from the hydrolysis by proteases like trypsin. Since trypsin is the major protease in human and predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine in human beings, we tested whether HP-modified proteins are sensitive to trypsin. Notably, treatment of HP-OVA with trypsin did not affect its anti-HIV-1 activity, indicating that the HP-modified lysine and arginine residues became resistant to trypsin. Notably, treatment of HP-OVA with trypsin, which predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, did not affect the anti-HIV-1 activity of HP-OVA, similar in SDS- PAGE, indicating that the protein containing the HP-modified lysine and arginine residues becomes resistant to trypsin.
An ideal microbicide candidate should be active against HIV-1 infection in the presence of human body fluids, such as seminal fluid or cervicovaginal fluid, because the topical microbicides will be applied intravaginally or intrarectally. In the present study, we tested the effects of seminal fluid and vaginal fluid simulant on anti-HIV activities of HP-OVA. The results indicate that the antiviral activities of HP-OVA are stable in the presence of those fluids, suggesting that HP-OVA should be active being used as a microbicide.
From 2000 to 2009, the failed clinical trials of several negatively charged polymeric microbicide candidates, such as Savvy (G31G), Cellulose sulfate (CS), 0.5% PRO 2000 gel and Carrageenan, made the research prospect of microbicide candidates is not optimistic. The main possible reason is that these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. The failures of these microbicide candidates have turned the focus on the next-generation microbicide candidates containing highly effective anti-HIV drugs in the present, such as TMC-120, UC781 and Tenofovir. One of the limitations of the anti-HIV drugs available in the market currently is the rapid emergence of HIV resistance. A combination of HP-OVA and other anti-HIV drug-based microbicide candidates with different target sites may have the following advantages:(i) maximizing antiviral efficacy, (ii) minimizing toxic effects and high cost due to combining dose reduction, (iii) fighting against diverse anti-HIV-1 drug mutations or delaying the emergence of HIV-1 resistance by using agents with different mechanisms and (iv) low price of production. Most importantly, HP-OVA is highly soluble and stable for long-term storage.
Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-OVA 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-OVA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-OVA 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-1 strains 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.
杀微生物剂是一类含有抗HIV成分的凝胶、乳脂、栓剂、药膜或海绵,在性交前置入阴道或肛门内,可预防艾滋病病毒和其它性传播疾病病毒的传播。杀微生物剂可通过直接灭活HIV、阻止HIV粘附及HIV侵入阴道或直肠粘膜内的靶细胞、抑制HIV在靶细胞内的复制等作用机理,保护健康人群在性行为时免受HIV感染。
理想的杀微生物剂应具备以下条件：广谱、安全、高效、在人体液中仍能保持较高抗病毒活性,对游离的病毒颗粒和细胞携带的病毒都应有抑制作用,对阴道、子宫颈或直肠粘膜上皮组织无损伤作用,对阴道共生菌群尤其是乳酸菌无抑制作用,具有耐酸、耐热、无色无味、价格低廉、易于使用等优点。基于这些特点,本文旨在寻找一类新型具特异性抑制病毒作用的候选杀微生物剂,用以预防和治疗HIV的性传播。
1995年,美国纽约血液中心的Neurath AR和姜世勃等人研究发现3-羟基-邻苯二甲酸酐(3-hydroxyphthalic anhydride,3-HP)修饰的p-乳球蛋白(p-lactoglobulin, P-LG),即3-HP-p-LG,可特异性抑制HIV的感染。而未经修饰的β-乳球蛋白及3-HP本身均无抑制HIV的活性,说明酸酐化学修饰将无活性的蛋白质转化成了具有抗HIV活性的病毒抑制剂。进一步研究还发现,3HP-β-LG具有极为广谱抗病毒活性,可有效对抗各种HIV-1实验室病毒株、HIV-1临床分离株、HIV-2和猿免疫缺陷病毒(simian immunodeficiency viruses, SIV)的感染。值得关注的是,3HP-β-LG还对其它性传播感染性病毒,如单纯疱疹病毒(HSV),沙眼衣原体(Chlamydia trachomatis)等,也具有较强的抑制活性。由于3HP-β-LG具有广谱、价廉、高效、稳定等特点,使其具备了被开发成杀微生物剂的条件。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-p-LG的研发受到了极大的影响,将其作为杀微生物剂研发的试验研究被迫终止。
本文旨在寻找一类新的非牛来源的蛋白来替代β-乳球蛋白,采用与3HP-B-LG相似的酸酐修饰方法,合成出新的酸酐修饰蛋白,通过对其抗病毒活性的分析,寻找出最具开发潜力的预防HIV性传播的候选杀微生物剂应用于临床。基于这一基本思路,本文选用了5种价格低廉、安全无毒的非牛奶来源的蛋白,包括：卵清蛋白OVA、兔血清白蛋白RSA、猪血清白蛋白PSA、冷水鱼皮明胶G-FS和猪皮明胶G-PS,作为被修饰对象,制备出一系列酸酐修饰蛋白,以期筛选出一类具有高效、低毒、价廉和稳定的候选杀微生物剂进行研发。
方法与结果：首先,选用3-羟基-邻苯二甲酸酐HP分别修饰上述5种动物蛋白,经化学合成方法合成5种不同的酸酐修饰蛋白(HP-OVA、HP-RSA、HP-PSA、HP-G-FS及HP-G-PS)。通过ELISA方法检测这5种酸酐修饰蛋白体外抗HIV-1 X4型(HIV-1ⅢB)及R5型(HIV-1Bal)病毒的活性。结果发现5种酸酐修饰蛋白具有不同程度的抗HIV-1病毒活性,其中抗病毒活性较高的为HP-OVA、HP-RSA和HP-PSA。比较分析这些酸酐修饰蛋白的抗病毒活性与被修饰蛋白的空间结构关系发现,OVA、RSA和PSA与β-乳球蛋白的结构相似,其空间结构均为球蛋白,而抗病毒活性较弱的被修饰蛋白G-FS和G-PS则是属于片层结缔状结构的明胶蛋白。由此可知,酸酐修饰的球蛋白的空间结构可能有助于其抗病毒活性的提高。3种动物蛋白(OVA、RSA和PSA)经HP酸酐修饰后虽都能转变成HIV抑制剂,我们选用卵清蛋白OVA作为后续研究蛋白,其主要原因是,OVA为鸡蛋清中的主要蛋白,占蛋清中总蛋白含量的60-65%,其来源广泛、价格低廉,极有可能被开发成具有自主知识产权、便宜、高效、应用简便的阴道用杀微生物剂,用于预防我国HIV的性传播。相比之下,其它两类蛋白均来源于血液,价格相对较高,且用药后具有潜在的感染其他病原体的危险性。
为了比较不同酸酐修饰OVA抗病毒活性的变化,本文选用3种酸酐：饱和酸酐(琥珀酸酐SU),不饱和酸酐(马来酸酐ML)及芳香族酸酐(3-羟基邻苯二甲酸酐HP)分别修饰OVA,得到3种酸酐修饰的卵清蛋白SU-OVA、ML-OVA和HP-OVA。通过比较这3种修饰蛋白的抗HIV-1活性,发现芳香族酸酐HP修饰蛋白后的产物HP-OVA的抗病毒活性最强,其次为不饱和酸酐修饰产物ML-OVA,最差为饱和酸酐修饰产物SU-OVA。由此可初步判定,酸酐中的苯环及不饱和烯键能明显提高酸酐修饰蛋白的抗病毒活性。综合上述结果,我们选用芳香族酸酐HP修饰的HP-OVA作为后续实验研究对象。
为优化HP-OVA的制备条件,我们用不同浓度的HP和不同的pH值反应体系,合成了的一系列不同的HP-OVA,并评价这些HP-OVA的抗病毒活性与蛋白末端碱性氨基酸残基被HP修饰比率的关系。结果发现,OVA蛋白末端的碱性氨基酸残基大部分能被HP所修饰,从而将无活性的OVA转变成具有较强抗HIV-1活性的病毒抑制剂HP-OVA。HP-OVA中赖氨酸和精氨酸残基的被修饰的比率越高,其抗HIV-1 X4和R5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-OVA抗病毒活性密切呈正相关。通过比较分析确定,HP-OVA化学修饰的最优合成条件为,酸酐HP终浓度40mM, pH值为8.5,在这一条件下,HP-OVA的蛋白末端赖氨酸被修饰的比率为99.86%,而精氨酸被修饰的比率为89.26%。
作为候选杀微生物剂,首先要具有广谱的抗病毒活性。经ELISA检测,HP-OVA具有极为广谱的抗病毒活性,能高效抑制多种HIV-1实验室病毒株包括HIV-1 X4型和R5型病毒感染,其IC5o均在低纳摩尔水平。同时,HP-OVA能高效抑制FDA批准的抗HIV药物,HIV进入抑制剂T20及逆转录酶抑制剂AZT耐受病毒株的感染,这为我们提供了一个新的治疗思路,即将HP-OVA与这些抗病毒药物合用,可能会产生取长补短的协同作用,这也是我们后续实验内容之一。
同时,HP-OVA也能高效地抑制各种HIV-1临床分离株的感染,包括A亚型-G亚型及O亚型(R5型或X4R5型),其IC50从0.011μM到0.578μM。特别是HP-OVA对HIV-1临床分离的A/E亚型病毒株感染具有很高的抑制活性,其IC5o仅为10 nM,而A/E亚型病毒株是性传播性疾病最为主要的传播病毒之
该病毒株在泰国较为流行,HP-OVA对该病毒株具有较好的抑制作用,预示着其在我国预防HIV性传播方面将会有较大的市场和应用前景。
另外,HP-OVA能有效抑制HIV-2感染,意味着HP-OVA也能被用在HIV-2病毒广泛流行的西非各国。与3HP-β-LG相似,HP-OVA也能对抗其他性传播性病原体HSV-2的感染。HP-OVA还能抑制SHIV和SIV的感染,由于SHIV和SIV病毒能够感染恒河猴,因此利用SHIV或SIV病毒阴道攻击恒河猴的动物模型,可先期评价HP-OVA在动物体内的抗病毒活性,为临床试验研究奠定了坚实的基础。
与目前临床试验失败的阴离子多聚体类候选杀微生物剂相比,HP-OVA的抗HIV-1病毒的活性,尤其是对HIV-1R5亚型病毒株的抑制作用明显增强,这极大地弥补了这些候选杀微生物剂的不足,从而使其具备被进一步开发的广阔前景。但HP-OVA为何能产生抗病毒活性,其作用机制如何,则是我们更为关注的问题。
通过time-of-addition、细胞-细胞融合、病毒-细胞融合及病毒细胞-细胞间的传播等实验结果分析可知,HP-OVA作用于HIV病毒的进入阶段,是一类HIV进入／融合抑制剂,其对HIV病毒感染靶细胞后期的复制过程无明显抑制作用。采用ELISA、SPR和流式细胞仪FCM分析还发现,HP-OVA通过与HIV包膜糖蛋白或病毒作用靶细胞上的CD4分子结合,干扰包膜糖蛋白与CD4分子的结合,从而抑制病毒与靶细胞膜的融合,阻止HIV的进入。通过ELISA和FN-PAGE方法,我们还发现较高浓度的HP-OVA对HIV跨膜糖蛋白gp41的六螺旋核心结构的形成也具有一定的抑制作用,从而抑制gp41介导的膜融合过程。这些结果说明,HP-OVA可能是一类多靶点的HIV进入抑制剂。
作为理想的杀微生物剂,除了高效之外,另一个必要条件是对人体安全无毒。体外安全性实验表明,HP-OVA对3种HIV病毒作用的T淋巴靶细胞及3种人正常阴道及子宫颈组织上皮细胞的细胞毒性均很低,其CC50的值远远大于其抑制HIV-1ⅢB病毒的IC50值,其选择系数(SI=CC50/IC50)范围为253到13066,说明HP-OVA体外对病毒靶细胞和组织上皮细胞基本无毒。阴道乳酸菌是一类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。作为外源性的杀微生物剂,不应该对阴道正常菌群的生长产生影响。因此,我们评价了HP-OVA对正常阴道乳酸菌生长是否有影响,结果发现22.4μM HP-OVA对17株正常人阴道乳酸杆菌的生长无明显抑制作用。
前述结果表明,HP-OVA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到至关重要的作用,HP-OVA用药是否会对人T淋巴细胞的功能产生影响呢?为此,我们首选观察了HP-OVA是否对正常人PBMCs及PHA刺激的PBMCs的增殖产生影响,结果证明,即使HP-OVA在浓度为100μM时,其对正常和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-OVA对正常和PHA刺激的PBMCs分泌细胞因子IFN-y功能的影响,结果同样证明HP-OVA对PBMCs的免疫功能无影响。这些结果初步表明,HP-OVA对CD4+T细胞的正常增殖和免疫功能,尤其是处于体内循环状态的T细胞不会产生有害的影响。
蛋白和多肽类药物,如T20,其主要的缺点之一为体内半衰期较短,极易被体内的蛋白酶水解。胰蛋白酶就是人体内重要的代谢蛋白酶之一,其在阴道菌群中也大量存在。胰蛋白酶裂解蛋白的主要位点是蛋白或多肽氨基酸末端的精氨酸或赖氨酸。由前述结果可知,OVA蛋白末端的碱性氨基酸,包括赖氨酸和精氨酸,绝大部分被HP酸酐所修饰,OVA蛋白末端氨基酸残基中胰蛋白酶的裂解位点有可能因酸酐修饰而发生改变。因此,我们检测了HP-OVA对胰蛋白酶的稳定性。结果表明,HP-OVA在胰蛋白酶的存在时,其抗病毒活性基本稳定,SDS-PAGE显示,蛋白结构也没有被酶所水解,而是保持相对稳定的分子量。
目前性传播已经成为HIV传播的主要途径,因此评估性传播中广泛存在的人体精液和宫颈阴道液对HP-OVA抗病毒活性的影响就显得至关重要了。本文结果可见,人体正常精液及阴道模拟液对HP-OVA的抗病毒活性并无显著影响。作为杀微生物剂制剂,样品的保存条件也不容忽视。我们将HP-OVA溶液保存于不同温度下,结果发现,不同温度对样品抗病毒活性无明显影响。
从2000年至2009年,几个候选杀微生物剂大规模临床Ⅲ期试验的相继失败,包括：Savvy (C31G), Cellulose sulfate (CS),0.5% PRO 2000凝胶及Carraguard等,使杀微生物剂研发前景不容乐观。而这些候选杀微生物剂临床试验失败的主原因之一被认为是由于它们对性传播过程中最为普遍的一类病毒株HIV-1 R5型病毒的抑制活性相对较低。为提高杀微生物剂的有效性,近年来研究的重点转向具有特异性、作用机制明确的第二代杀微生物剂。目前临床上研发的第二代候选杀微生物剂主要为逆转录酶抑制剂,如TMC-120, UC781和Tenofovir,但它们也有相应的缺陷,如极易诱发产生病毒耐药株等。HP-OVA具备广谱、高效、价格低廉、易于制备及安全稳定等特点,且对大部分药物产生的耐药株有较好的抑制活性。HP-OVA为多靶点的HIV进入抑制剂,故其临床应用可能不易产生耐药株。因此,本文选用一系列可能被发展成为候选杀微生物剂的抗病毒药物,包括HIV进入抑制剂和逆转录酶抑制剂,与HP-OVA合用,观察HP-OVA与各药单用及合用后的协同作用效果。结果发现,HP-OVA与各药合用时,均能产生不同程度的协同作用效果。HP-OVA与第二代候选杀微生物剂合用具有以下优点：1)增强抗病毒活性,扩大抗病毒谱；2)降低毒副作用,减少用药剂量；3)多靶点用药,延缓病毒耐药株的产生；4)降低药物成本,优化保存条件。
结论：酸酐修饰卵清蛋白HP-OVA具有高效广谱的抗病毒活性,能有效对抗各种HIV-1实验室病毒株和临床分离株的感染,同时还具有抑制HIV-2、SHIV、SIV及HSV-2的作用；机制研究表明,HP-OVA通过与HIV包膜糖蛋白或病毒作用靶细胞上的CD4分子结合,干扰包膜糖蛋白与CD4分子的结合,从而抑制病毒与靶细胞膜的融合,阻止HIV的进入；体外安全性及稳定性试验证明,HP-OVA对病毒作用靶细胞和阴道正常细胞安全无毒,其对阴道正常菌群的分泌及CD4+T淋巴细胞的增殖和免疫功能均无明显影响,胰蛋白酶及人体体液对其活性影响不大；其蛋白来源广泛、价格低廉,具备发展成理想的杀微生物剂的基本条件,可作为预防HIV性传播的杀微生物剂进行研发；HP-OVA能和具有开发成杀微生物剂潜能的抗病毒药物合用,具有提高疗效,减少药物毒副作用的协同作用效果,这为寻找新的预防HIV性传播治疗策略提供了较好的理论基础,也为杀微生物剂的临床开发提供新的思路。
Objection:Despite extraordinary advances in the development of prevention and therapeutic strategies against human immunodeficiency virus (HIV) infection, HIV/AIDS continues to spread at an alarming rate worldwide. There are approximately 7,400 new infections and over 5,500 new deaths resulting from AIDS each day. There are three main transmission routes for HIV, including by blood, mother to child and unprotected sex. Unprotected sex is the primary route for humans now, especially for females in many developing countries, to acquire HIV/AIDS. Therefore, development of female-controlled topical microbicides is urgently needed.
Micribicides are gels, creams, foams, or suppositories, which can be applied in vagina or rectum before sex to prevent sexual transmission of HIV. These products are designed to protecting HIV infection by killing HIV, inhibiting HIV entry target cells or HIV replication in the cells.
An ideal microbicide should be broad antiviral activity, safe, highly effective, affordable, stable in human fluids, and easy to use. It should be safe to human tissues and to vigina bacteria. Microbicides should also be cheap to be used in many developing countries. Based on these characters, our group is searching for some new anti-HIV microbicide candidates for preventing HIV sexually transmission.
In 1995, our group of New York Blood Center demonstrated that bovine B-lactoglobulin, a protein present in milk and whey, modified by 3-hydroxyphthalic anhydride (3HP-B-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1 and HSV-2). Nether non-modified B-lactoglobulin nor 3-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 of 3HP-B-LG as a microbicide.
Therefore, in the present study, we sought to replace bovine proteins with chemically modified animal proteins of non-bovine origin as new anti-HIV microbicide candidates. All of the non-bovine animal proteins including chicken ovalbumin (OVA), rabbit serum albumin (RSA), porcine serum albumin (PSA), gelatin from cold water fish skin (G-FS), gelatin from porcine skin (G-PS) were modified by 3-hydroxyphthalic anhydride (HP), using the same method and the same conditions as 3HP-B-LG.
Methods and Results:After extensive screening, we found that several HP-modified non-bovine-origin proteins exhibited inhibitory activity (HP-OVA, HP-RSA, HP-PSA, HP-G-FS, HP-G-PS) against infection by HIV-1 X4 (HIV-1ⅢB) and R5 (HIV-1BaL) viruses. By analyzing the structure of the proteins found to possess antiviral activity, OVA, RSA and PSA were found to have representative globulins identical to bovineβ-lactoglobulin. By contrast, the gelatins used in this study are derived from collagens, which had different structure and conformation. The absence of anti-HIV activities of these modified proteins indicated that HIV blocking abilities might not be solely dependent on the modified lysine or arginine but also on the protein conformation. Thus, the presence of specific globular structures might play an important role in the anti-HIV activity of OVA, RSA and PSA. y evaluating the anti-HIV activities of these modifications and the characteristics of proteins used in the reaction, we found that HP-modified chicken ovalbumin (HP-OVA) was the most promising anti-HIV inhibitor among these modified proteins. Chicken ovalbumin (OVA) is the main protein in egg white, making up 60-65% of the total protein. Since OVA is one of the most abundant proteins consumed by people worldwide and is a generally recognized as a safe (GRAS) protein, HP-modified OVA has great potential for further development as an effective, safe and affordable microbicide.
To search for alternate anhydrides as chemical modifiers of OVA, we selected two other anhydrides, succinic anhydride (SU) and maleic anhydride (ML), for the chemical modification. We compared the efficiency of three different anhydrides, including ML, SU, as well as HP, for the chemical modification of OVA. All three anhydrides (SU, ML and HP) were sufficiently potent to convert OVA into an effective anti-HIV agent. By compared the anti-HIV-1 activity of those three anhydride OVAs, HP-OVA and ML-OVA demonstrated more efficacy than SU-OVA in blocking HIV-1 infection, especially the sexually transmitted R5 virus. It is the chemical structure of anhydrates that accounts for the effect of different anhydride OVA modifications on HIV inhibitory activities. Specifically, the only difference between maleic and succinic anhydride was the double bond between C3 and C4 in maleic anhydride, which led to the stronger inhibition abilities of ML-OVA over those of SU-OVA on HIV infection.3-hydroxyphthalic anhydride has a hydrophobic aromatic group, leading to the most potent anti-HIV activity. These findings suggest that the aromatic and unsaturated structure in anhydrides might contribute to the difference in antiviral activities of these modified OVAs.
For confirming the optimal condition, we prepared a series of HP-OVA by changing the different HP concentration and different pH values. Here we found that the percentage of the HP-modified and unmodified lysine and arginine residues in OVA was dependent on the concentration of HP and pH of the reaction system and was correlated with the anti-HIV-1 activity of HP-OVA. These results suggested that the modified amino acid residues play an important role in mediating the antiviral activity. We selected 40 mM concentration of HP and pH 8.5 as the optimal condition for preparation of the HP-OVA for the subsequent studies. Under such condition, HP-OVA had 99.86% and 89.26% of the lysine and arginine residues modified by HP, respectively.
By ELISA, HP-OVA effectively inhibited infection by laboratory-adapted HIV-1 strains, including X4 and R5 viruses, with IC50 in the nM range. HP-OVA is highly effective in inhibiting the infection of the primary R5 viruses with distinct genotypes and phenotypes, with IC50 from 0.011μM to 0.578μM. HP-OVA is more effective against the predominant HIV-1 subtypes A, B, and C. Particularly, HP-OVA is highly potent against the primary HIV-1 isolated in Thailand,92TH009 (subtype A/E, R5) with IC50 about 10 nM, while it was reported that the HIV-1 subtype A/E R5 virus was preferentially sexually transmitted. Those results suggest that HP-OVA have a great potency to be used as a microbicide candidate in the world, especially in our country.
HP-OVA is also effective in inhibiting HIV-2 infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2 is prominent. Our studies also showed that HP-OVA could potently inhibit infection by SHIV and SIV. Since both SHIV and SIV can be used for infection of rhesus macaques, HP-OVA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SHIV or SIV infection through vaginal challenge.
Compared with several negatively charged polymeric microbicide candidates failed in clinical trials, most of these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. However, this is not the case for HP-OVA since it is almost equally effective against both X4 and R5 viruses. Next, we try to find the mechanism of HP-OVA for inhibiting the HIV infection.
By using time-of-addition, cell-cell fusion, and cell-to-cell transmission assays, we demonstrated that HP-OVA is an HIV entry/fusion inhibitor since it exhibited significantly decreased inhibitory activity when it was added after HIV-1 infection and it showed potent inhibitory activity on cell-to-cell fusion mediated by X4 virus and cell-to-cell transmission of R5 virus. Using ELISA, SPR and FCM assay, HP-OVA could block the binding of the HIV-1 Env surface subunit gp120 (from both X4 and R5 viruses) or an anti-CD4 antibody to sCD4, the primary receptor for HIV, resulting in inhibition of interaction between gp120 and CD4. Furthermore, we demonstrated that HP-OVA could block the formation of the fusion-active gp41 six-helix bundle. These results suggest that HP-OVA inhibit HIV-1 entry/fusion through multiple mechanisms of action by interacting with both gp120 and gp41 as well as CD4 via the negatively charged residues of HP-OVA.
The failure of microbicide candidates clinical trials warned us that the safety evaluation of a microbicide candidate should be carried out as early as possible. Here we first assessed the potential cytotoxicity of HP-OVA on three well-characterized cell lines derived from the human vaginal and cervical epithelium and three human T immune cell lines as well as PBMCs, which were used for evaluation of the in vitro anti-HIV-1 activity of HP-OVA. The results showed that HP-OVA had low cytotoxicity to all tested cells. Its selectivity index (SI=CC50/IC50) ranged from 253 to 13,066, indicating that HP-OVA appears to be safe in vitro. More extensive animal studies to evaluate its in vivo toxicity will be carried out in the future. Vaginal lactic acid bacteria are not be affected by HP-OVA, even in the high concentration in 22.4μM.
Since HP-OVA can bind to sCD4 while the CD4 molecule on the T lymphocytes plays an important role in T cell activation, one of the concerns is whether HP-OVA affects the function of CD4+ T cells or induces T cell anergy, a status of the lymphocyte that is functionally inactivated following an antigen stimulation. Our studies demonstrated that proliferation of T lymphocytes in human PBMCs stimulated with PHA was not significantly affected by HP-OVA at the concentration as high as 100μM. Further studies showed that HP-OVA had no significant effect on the productions of IFN-y by PBMCs with or without PHA stimulation. Those results suggest that HP-OVA may not have deleterious effects on the function of CD4+ T cells, especially for those circulating in the human body. But we cannot exclude the possibility that long-term use of CD4 blockers topically may suppress the function of CD4+ immune cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-OVA on the mucosal immune system is warranted.
The disadvantage of protein/peptide drugs, such as T20, is their short half life resulting from the hydrolysis by proteases like trypsin. Since trypsin is the major protease in human and predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine in human beings, we tested whether HP-modified proteins are sensitive to trypsin. Notably, treatment of HP-OVA with trypsin did not affect its anti-HIV-1 activity, indicating that the HP-modified lysine and arginine residues became resistant to trypsin. Notably, treatment of HP-OVA with trypsin, which predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, did not affect the anti-HIV-1 activity of HP-OVA, similar in SDS- PAGE, indicating that the protein containing the HP-modified lysine and arginine residues becomes resistant to trypsin.
An ideal microbicide candidate should be active against HIV-1 infection in the presence of human body fluids, such as seminal fluid or cervicovaginal fluid, because the topical microbicides will be applied intravaginally or intrarectally. In the present study, we tested the effects of seminal fluid and vaginal fluid simulant on anti-HIV activities of HP-OVA. The results indicate that the antiviral activities of HP-OVA are stable in the presence of those fluids, suggesting that HP-OVA should be active being used as a microbicide.
From 2000 to 2009, the failed clinical trials of several negatively charged polymeric microbicide candidates, such as Savvy (G31G), Cellulose sulfate (CS), 0.5% PRO 2000 gel and Carrageenan, made the research prospect of microbicide candidates is not optimistic. The main possible reason is that these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. The failures of these microbicide candidates have turned the focus on the next-generation microbicide candidates containing highly effective anti-HIV drugs in the present, such as TMC-120, UC781 and Tenofovir. One of the limitations of the anti-HIV drugs available in the market currently is the rapid emergence of HIV resistance. A combination of HP-OVA and other anti-HIV drug-based microbicide candidates with different target sites may have the following advantages:(i) maximizing antiviral efficacy, (ii) minimizing toxic effects and high cost due to combining dose reduction, (iii) fighting against diverse anti-HIV-1 drug mutations or delaying the emergence of HIV-1 resistance by using agents with different mechanisms and (iv) low price of production. Most importantly, HP-OVA is highly soluble and stable for long-term storage.
Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-OVA 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-OVA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-OVA 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-1 strains 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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84. Lederman MM, Jump R, Pilch-Cooper HA, et al. Topical application of entry inhibitors as "virustats" to prevent sexual transmission of HIV infection. Retrovirology 2008,5:116.
85. Fichorova RN, Zhou F, Ratnam V, et al. Anti-human immunodeficiency virus type 1 microbicide cellulose acetate 1,2-benzenedicarboxylate in a human in vitro model of vaginal inflammation. Antimicrob Agents Chemother 2005,49:323-335.
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88. Di TA, Saletti G, Pizza M, et al. Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant. Infect Immun 1996,64:974-979.
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94. Veazey RS, Klasse PJ, Schader SM, et al. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature 2005,438:99-102.
95. Wang T, Zhang Z, Wallace OB, et al. Discovery of 4-Benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806):A Novel HIV-1 Attachment Inhibitor That Interferes with CD4-gp120 Interactions. JMed Chem 2003,46:4236-4239.
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97. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006,58:621-681.