人呼吸道合胞病毒活疫苗研究进展

人呼吸道合胞病毒是引起婴幼儿支气管炎和肺炎的主要原因,也可导致免疫缺陷病人及老年人群显著发病和死亡.人呼吸道合胞病毒疫苗已被世界卫生组织(World Health Organization,WHO)列为全球最优先发展的疫苗之一.经过50多年的研究,尤其是随着重组技术和反向遗传学的出现,对RSV疫苗的研究取得了重要进展,...     

Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.     

Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.     

Growth of Respiratory Syncytial Virus in Suspension Cell Culture

Respiratory syncytial virus, Burnett strain, adsorbed efficiently and grew to high titers in suspension cultures of HEp-2 and MA-160 cells. Our results compared favorably with previous experience with the growth of respiratory syncytial virus in monolayer cell cultures. The use of suspension cell cultures provides a convenient and simple procedure for producing high-titering respiratory syncytial virus pools.     

Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

树鼩呼吸道合胞病毒感染动物模型的建立

探讨用灵长类动物树鼠句建立呼吸道合胞病毒(RSV)感染动物模型的可行性。28只树鼠句随机分为7组,每组4只,鼻内滴入106PFU RSV,感染后每24h检测1组。另取7只树鼠句鼻内滴入100μl Hep-2细胞培养液,滴鼻后每24h检测1只作对照。无菌取树鼠句肺组织进行病毒分离、空斑形成实验检测病毒滴度、病理检查和RT-PCR检测肺组织内RSV mRNA表达。结果显示:树鼠句感染RSV后,无明显呼吸道感染症状;树鼠句肺组织匀浆接种于Hep-2细胞上,第3、4、5实验组出现细胞病变效应(CPE);树鼠句感染RSV后肺部病理改变在3~7d较为明显,主要为间质改变;在106PFU感染浓度下树鼠句肺组织内RSV处于低水平复制,复制的高峰期在3~5d,峰值在第4d。提示:树鼠句可以用来建立RSV感染的动物模型。     

Identification of a gp41 Core-Binding Molecule with Homologous Sequence of Human TNNI3K-Like Protein as a Novel Human Immunodeficiency Virus Type 1 Entry Inhibitor

Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4⁺ cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus, and its envelope protein (Env) complex controls the key processes by which HIV-1 delivers its replicative material into target cells. Specifically, the Env surface subunit, gp120, binds the cellular receptor CD4 and a coreceptor, CCR5 or CXCR4, which triggers conformational changes of the transmembrane subunit, gp41 (8). The N-terminal heptad repeat (NHR) in the gp41 ectodomain interacts with its C-terminal heptad repeat (CHR) to form a trimer of hairpins, or six-helix bundle (6-HB; also known as the gp41 fusion core) (38, 51), which brings the viral and target cell membranes into close proximity and promotes membrane fusion (3, 51). Therefore, the gp41 6-HB core plays an important role in viral fusion and may serve as an attractive target for the development of HIV fusion/entry inhibitors (20).In the early 1990s, a number of peptides derived from the gp41 NHR and CHR regions were discovered to exhibit highly potent anti-HIV-1 activity by binding to the corresponding region of gp41 at the fusion-intermediate state (22, 23, 38, 52, 53) and blocking gp41 6-HB core formation (4, 9, 32, 47). One of the CHR-peptides, T-20 (generic name, enfuvirtide; brand name, Fuzeon), was licensed by the FDA as the first member of a new class of anti-HIV drugs, the HIV fusion inhibitors (33, 53). Although T-20 is very effective in inhibiting infection by a broad spectrum of HIV-1 strains, especially those resistant to current antiretroviral therapies (26), T-20 itself also can easily induce drug resistance in T-20-treated patients, resulting in virologic failure (36, 46, 50, 55). Therefore, it is essential to identify and develop novel HIV-1 fusion inhibitors having a mechanism of action or target different from that for T-20 and with improved drug resistance profiles.Here, we sought to screen a human bone marrow cDNA library in a yeast two-hybrid screening assay using the recombinant soluble gp41 ectodomain (rsgp41e) as the bait in hopes of identifying a novel HIV fusion inhibitor with sequence homology to a human protein and low immunogenicity to humans to avoid its rapid clearance by specific human antibodies (1). We identified a 32-mer peptide, designated P20, with sequence homology to human troponin I type 3 interacting kinase (TNNI3K)-like protein. P20 could specifically bind to the gp41 6-HB core and strongly blocked HIV-1 Env-mediated membrane fusion. It potently inhibited infection by a number of laboratory-adapted HIV-1 strains, including T-20-resistant variants, and a broad spectrum of primary HIV-1 isolates. These results suggest that P20 has the potential to be developed further as a novel anti-HIV-1 therapeutic and can be used as a probe to study the role of the HIV-1 gp41 6-HB core in the membrane fusion process.     

Fluorescent Cell Counting as an Assay Method for Respiratory Syncytial Virus

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log(10) units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells.     

Characterization of Ebola Virus Entry by Using Pseudotyped Viruses: Identification of Receptor-Deficient Cell Lines

Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 × 10⁶ infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.     

Temperature-sensitive Mutants of Respiratory Syncytial Virus

Four conditional-lethal temperature-sensitive mutants of RS virus were detected among the progeny of 454 plaques derived from virus grown in the presence of 10(-4)m 5-fluorouridine. These mutants were stable (reversion frequency, 10(-5.0) or less and failed to produce plaques at 38 or 39 C. Plaquing efficiency was depressed 100-fold or more at 37 C. Variable suppression of growth at the restrictive temperature of 39 C was observed, ranging from 16-fold to complete suppression. The temperature-sensitive defect of three of the mutants appeared to affect functions which were expressed late in the replicative cycle. One of the mutants produced atypical nonsyncytial plaques.     

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC₅₀ values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.     

Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)₂ form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.     

抗呼吸道合胞病毒融合蛋白单链抗体的筛选及初步鉴定

目的：从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法：以RSV F蛋白为靶抗原,通过“吸附-洗涤-洗脱-扩增”过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western及Dot blot分析。结果：经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明为单链抗体。结论：利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。