广谱趋化因子受体结合物-vMIP-Ⅱ的体内抗SIV功能研究

病毒巨噬细胞炎症蛋白Ⅱ(vMIP-Ⅱ)是一种广谱趋化因子受体拮抗剂,它所拮抗的趋化因子受体被认为是不同的人免疫缺陷病毒株(Human immunodeficiency virus,HIV)进入靶细胞的辅受体.虽然理论上vMIP-Ⅱ是一个广谱的HIV抑制剂,但vMIP-Ⅱ的抗HIV感染作用却少有报道,特别是体内研究.本研究利用一个有效的SIV-mac251感染食蟹猴模型来评价vMIP-Ⅱ的体内抗HIV感染作用,结果显示vMIP-Ⅱ能够有效地并呈剂量依赖性地降低食蟹猴血浆病毒载量,同时对宿主免疫功能具有保护作用.这些结果表明vMIP-Ⅱ是一种有效的抗HIV物质,可以作为一类新型的抗HIV先导药物,也为研发靶向病毒进入的新药提供了进一步的理论支持.     

广谱趋化因子受体结合物-vMIP—Ⅱ的体内抗SIV功能研究

病毒巨噬细胞炎症蛋白Ⅱ（vMIP-Ⅱ）是一种广谱趋化因子受体拮抗剂,它所拮抗的趋化因子受体被认为是不同的人免疫缺陷病毒株（Human immunodeficiency virus,HIV）进入靶细胞的辅受体。虽然理论上vMIP—Ⅱ是一个广谱的HIV抑制剂,但vMIP—Ⅱ的抗HIV感染作用却少有报道,特别是体内研究。本研究利用一个有效的SIV—mac251感染食蟹猴模型来评价vMIP-Ⅱ的体内抗HIV感染作用,结果显示vMIP-Ⅱ能够有效地并呈剂量依赖性地降低食蟹猴血浆病毒载量,同时对宿主免疫功能具有保护作用。这些结果表明vMIP—Ⅱ是一种有效的抗HIV物质,可以作为一类新型的抗HIV先导药物,也为研发靶向病毒进入的新药提供了进一步的理论支持。     

慢病毒载体感染成年食蟹猴骨髓间充质干细胞

骨髓间充质干细胞(Mesenchymal stem cells,MSCs)具有增殖和多向分化潜能,临床应用广泛,近年来备受关注。另一方面,MSCs易于转导和表达外源基因,是理想的基因工程细胞。非人灵长类(NHPs)和人类具有非常相近的遗传背景,NHPs模型在评价药物疗效和移植治疗等方面具有不可替代的价值。本研究采用密度梯度离心法分离成年食蟹猴骨髓单核细胞(Marrow mononuclear cells,MNCs),贴壁培养MSCs。同时构建表达绿色荧光蛋白(Green fluorescent protein,GFP)的慢病毒载体,感染成年食蟹猴MSCs。结果显示,体外培养的成年食蟹猴MSCs均感染猴泡沫病毒(Simian foamy virus,SFV),体外培养成年食蟹猴MSCs必须添加抗病毒药物Tenofovir。但由于食蟹猴MSCs感染SFV,以及培养中添加了抗病毒药物Tenofovir,慢病毒载体的感染效率明显降低(10%)。本研究通过停用抗病毒药,在细胞复苏后6d转染慢病毒,可大幅提高慢病毒的感染效率(50%)。为成年食蟹猴MSCs作为基因工程细胞应用于实验和临床研究提供了技术保证。     

GDNF慢病毒载体感染食蟹猴骨髓间充质干细胞

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是基因工程和细胞治疗的种子细胞之一,本研究利用含胶质源性神经营养因子(glial cell derived neurotrophic factor,GDNF)基因的慢病毒载体感染成年食蟹猴MSCs,探讨转染后GDNF在MSCs中的体外表达水平及其影响因素。首先,通过密度梯度离心法分离食蟹猴骨髓单核细胞(marrow mononuclear cells,MNCs),体外培养食蟹猴MSCs。同时构建表达GDNF的慢病毒载体,并感染食蟹猴MSCs,分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法,测定感染不同拷贝数病毒和不同转染组细胞GDNF的蛋白分泌水平和基因表达水平。实验结果显示,表达GDNF基因的慢病毒载体成功转染成年食蟹MSCs,体外培养的MSCs持续表达分泌GDNF。感染慢病毒的拷贝数可以影响GDNF分泌水平,相同条件下感染拷贝数越高,GDNF分泌量越多,其基因表达水平越高。     

vMIP-Ⅱ--一种广谱的趋化因子受体拮抗蛋白

人疱疹病毒8型编码的一种趋化因子类似物(vMIP-Ⅱ)能与大量CC类和CXC类细胞因子受体高度亲和性结合,通过拮抗Ca^2 短暂迅速内流引起的信号传导,阻断趋化因子受体的趋化作用。vMiP-Ⅱ对趋化因子受体的广谱拮抗能力为开发广谱的抗人类免疫缺陷病毒药物、在非全身免疫抑制条件下治疗移植免疫排斥反应以及治疗其他炎性疾病引入了新的途径。     

SARS动物模型的研究

利用分离的SARS CoV毒株BJ 0 1,经滴鼻等途径感染大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等 5个种属的动物 ,筛选对SARS易感的小动物。在此基础上 ,选择食蟹猴和恒河猴进行SARS的人工感染实验 ,评价其作为SARS动物模型的可能性。结果表明 ,大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等动物对SARS均不易感 ,感染后未观察到任何的临床及病理学改变 ,不过从感染 2周后的大鼠和豚鼠的肺和咽等组织样本中检测到了的特异的核酸 ,提示SARS CoV能够在这两种动物的体内复制。从感染猴子的分泌物和脏器中分离出了病毒 ,证明SARS CoV也能够在猴子体内复制。临床和病理组织学检查结果显示 ,SARS病毒接种食蟹猴和恒河猴后 ,可以引起所有实验猴发生间质性肺炎 ,其病理学改变与人类感染SARS病毒后肺部病变近似 ,但病变的严重程度比较人类的轻得多 ,除此之外无任何其它的明显的临床表现及组织病理学改变 ,按照动物模型的指标判断食蟹猴和恒河猴并不是SARS的理想动物模型 ,不过在目前尚没有更理想的动物模型情况下 ,以间质性肺炎为病理学检查指标 ,恒河猴和食蟹猴可以作为评价抗SARS药物和疫苗的模型动物     

中国圈养食蟹猴TrimCyp基因频率

TRIM 5α在绝大部分的旧大陆猴中扮演抗逆转录病毒的角色,能够限制HIV-1的活性。TRIMCyp融合基因是继TRIM 5α后的另一个抗HIV-1因子研究热点。旧大陆猴的TRIMCyp融合基因是由CypA假基因cDNA序列以逆转录转座的方式插入至TRIM5基因的3’非翻译区形成,而且TRIMCyp融合基因在不同灵长类动物中具有地域、基因频率、基因型以及抗逆转病毒效应的差异。虽然食蟹猴TRIMCyp基因的频率在东南亚几个国家或地区已经被初步调查,但是,中国大陆食蟹猴养殖场的TRIMCyp基因频率还没有明确阐明。该研究对中国5个省11个养殖场共1594个食蟹猴(Macaca fascicularis)繁殖种群随机样本的TRIMCyp基因频率进行了筛查研究,发现各场频率略有差异,从7.65%～19.79%不等,显著低于已报道的菲律宾、马来西亚和印度尼西亚来源食蟹猴的TRIMCyp基因频率(34.85%~100%)。该原因可能是由于后者是建立于1978年的封闭群。对带有TRIMCyp融合基因的个体CypA测序发现带有NE单倍型的食蟹猴个体很少,NE单倍型频率(4.93%)显著低于东南亚三个国家食蟹猴的NE单倍型频率(11.1%～14.3%)纯合子。该研究为进一步开展食蟹猴HIV-1动物模型和发病机制提供了基础信息。     

CD4-CCR5工程类重组二价抗体的慢病毒包装及制备

人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染与白细胞分化抗原分子CD4和趋化因子受体分子CCR5有着紧密的联系。为了研制可与HIV病毒特异性结合,并能有效预防和治疗HIV感染的抗HIV基因工程二价类重组药物,将人抗体重链恒定区IgG与CD4相连接,CCR5与人抗体轻链恒定区连接,分别构建慢病毒质粒,共转染并筛选,得到高效稳定表达的细胞株。重组抗体经分子生物学及细胞免疫学检测,证实具有抗HIV病毒感染的功能,有一定的经济价值。     

人工驯养条件下食蟹猴B病毒抗体水平变化规律研究

目的研究人工驯养条件下食蟹猴B病毒抗体水平变化规律,便于有效控制自繁食蟹猴的BV感染率。方法随机选取409只对不同月龄自繁食蟹猴,采用BVELISA法进行BV抗体监测。结果新生仔猴刚出生时均携带不同程度的BV抗体,但随着月龄的增加,BV抗体水平开始下降,至5月龄时BV抗体阳性率降至最低（12．3％）,之后BV抗体水平逐渐升高。结论人工驯养条件下食蟹猴B病毒抗体水平呈由高到低再升高的趋势,5月龄时断奶可最大限度地获得BV抗体阴性猴。     

抗人类免疫缺陷病毒1型广谱中和抗体的研究进展

现行抗反转录病毒治疗药物的联合应用可有效抑制艾滋病进程并显著延长患者寿命,但由于人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)潜伏库的存在,艾滋病迄今尚无法治愈。近年发现抗HIV广谱中和抗体能有效降低患者体内病毒载量并延缓疾病进程,为研发艾滋病疫苗和治愈策略带来了曙光,尤其是序贯免疫策略的使用极大推进了广谱中和抗体的开发和应用进程。2018年,美国食品药品管理局(Food and Drug Administration,FDA)批准了第1个临床应用的广谱中性单克隆和抗体,无疑为抗HIV单克隆抗体药物的研发注入了一支强心剂。本文围绕近年来抗HIV广谱中和抗体的研究进展进行综述,探讨未来广谱中和抗体研发面临的挑战。     

The antiviral protein cyanovirin-N: the current state of its production and applications

Human immunodeficiency virus (HIV)/AIDS continues to spread worldwide, and most of the HIV-infected people living in developing countries have little or no access to highly active antiretroviral therapy. The development of efficient and low-cost microbicides to prevent sexual transmission of HIV should be given high priority because there is no vaccine available yet. Cyanovirin-N (CVN) is an entry inhibitor of HIV and many other viruses, and it represents a new generation of microbicide that has specific and potent activity, a different mechanism of action, and unusual chemicophysical stability. In vitro and in vivo antiviral tests suggested that the anti-HIV effect of CVN is stronger than a well-known gp120-targeted antibody (2G12) and another microbicide candidate, PRO2000. CVN is a cyanobacteria-derived protein that has special structural features, making the artificial production of this protein very difficult. In order to develop an efficient and relatively low-cost approach for large-scale production of recombinant CVN to satisfy medical use, this protein has been expressed in many systems by trial and error. Here, to summarize the potential and remaining challenges for the development of this protein into an HIV prevention agent, the progress in the structural mechanism determination, heterologous production and pharmacological evaluation of CVN is reviewed.     

Crystal structure and structural mechanism of a novel anti-human immunodeficiency virus and D-amino acid-containing chemokine

Chemokines and their receptors play important roles in normal physiological functions and the pathogeneses of a wide range of human diseases, including the entry of human immunodeficiency virus type 1 (HIV-1). However, the use of natural chemokines to probe receptor biology or to develop therapeutic drugs is limited by their lack of selectivity and the poor understanding of mechanisms in ligand-receptor recognition. We addressed these issues by combining chemical and structural biology in research into molecular recognition and inhibitor design. Specifically, the concepts of chemical biology were used to develop synthetically and modularly modified (SMM) chemokines that are unnatural and yet have properties improved over those of natural chemokines in terms of receptor selectivity, affinity, and the ability to explore receptor functions. This was followed by using structural biology to determine the structural basis for synthetically perturbed ligand-receptor selectivity. As a proof-of-principle for this combined chemical and structural-biology approach, we report a novel D-amino acid-containing SMM-chemokine designed based on the natural chemokine called viral macrophage inflammatory protein II (vMIP-II). The incorporation of unnatural D-amino acids enhanced the affinity of this molecule for CXCR4 but significantly diminished that for CCR5 or CCR2, thus yielding much more selective recognition of CXCR4 than wild-type vMIP-II. This D-amino acid-containing chemokine also showed more potent and specific inhibitory activity against HIV-1 entry via CXCR4 than natural chemokines. Furthermore, the high-resolution crystal structure of this D-amino acid-containing chemokine and a molecular-modeling study of its complex with CXCR4 provided the structure-based mechanism for the selective interaction between the ligand and chemokine receptors and the potent anti-HIV activity of D-amino acid-containing chemokines.     

A novel peptide antagonist of CXCR4 derived from the N-terminus of viral chemokine vMIP-II

The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus is unique among all known chemokines in that vMIP-II shows a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus type 1 (HIV-1). To elucidate the mechanism of the promiscuous receptor interaction of vMIP-II, synthetic peptides derived from the N-terminus of vMIP-II were studied. In contrast to the full-length protein that recognizes both CXCR4 and CCR5, a peptide corresponding to residues 1-21 of vMIP-II (LGASWHRPDKCCLGYQKRPLP) was shown to strongly bind CXCR4, but not CCR5. The IC(50) of this peptide in competing with CXCR4 binding of (125)I-SDF-1alpha is 190 nM as compared to the IC(50) of 14.8 nM of native vMIP-II in the same assay. The peptide selectively prevented CXCR4 signal transduction and coreceptor function in mediating the entry of T- and dual-tropic HIV-1 isolates, but not those of CCR5. Further analysis of truncated peptide analogues revealed the importance of the first five residues for the activity with CXCR4. These results suggest that the N-terminus of vMIP-II is essential for its function via CXCR4. In addition, they reveal a possible mechanism for the distinctive interactions of vMIP-II with different chemokine receptors, a notion that may be further exploited to dissect the structural basis of its promiscuous biological function. Finally, the potent CXCR4 peptide antagonist shown here could serve as a lead for the development of new therapeutic agents for HIV infection and other immune system diseases.     

Effect of the extent of thiolation and introduction of phosphorothioate internucleotide linkages on the anti-HIV activity of Suligovir [(s4dU)35

Suligovir is a 35-mer homo-oligonucleotide, containing exclusively 4-thio deoxyuridylate, proved to be a potent inhibitor of HIV entry. In this paper, we described the effect of extent of thiolation and the introduction of nuclease-resistant phosphorothioate linkages on the anti-HIV activity of Suligovir. We found that the decreased thiolated nucleotide content decreases the anti-HIV potency of the compound and the introduction of phosphorothioate linkages does not improve its antiviral activity.     

Development of HIV entry inhibitors targeted to the coiled-coil regions of gp41

The discoveries that synthetic peptides corresponding to the N- and C-terminal heptad repeat (HR) regions of gp41 have potent anti-HIV activity opened a new avenue to identification of small molecule HIV entry inhibitors targeted to the HIV gp41 coiled-coil regions. Based on the structural information of the HIV gp41 core, three distinct approaches to develop small molecule anti-HIV agents have been reported. Each of these approaches has specific advantages, which will have complementary effects on the design of new strategies for identification of more potent HIV entry inhibitors. It is expected that novel antiviral drugs targeted to the HIV gp41 coiled-coil regions will be developed in the near future for the chemotherapy and/or prophylaxis of HIV infection and AIDS.     

CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.     

The virus-encoded chemokine vMIP-II inhibits virus-induced Tc1-driven inflammation

The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8(+) T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8(+) T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.     

Biophysical property and broad anti-HIV activity of albuvirtide, a 3-maleimimidopropionic acid-modified peptide fusion inhibitor

Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B', CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.     

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.