The larger attachment glycoprotein of respiratory syncytial virus produced in primary human bronchial epithelial cultures reduces infectivity for cell lines

Respiratory syncytial virus (RSV) infects the upper and lower respiratory tracts and can cause lower respiratory tract infections in children and elders. RSV has traditionally been isolated, grown, studied and quantified in immortalized cell lines, most frequently HEp-2 cells. However, in vivo RSV infection is modeled more accurately in primary well differentiated human bronchial epithelial (HBE) cultures where RSV targets the ciliated cells and where the putative RSV receptor differs from the receptor on HEp-2 cells. The RSV attachment (G) glycoprotein in virions produced by HEp-2 cells is a highly glycosylated 95 kDa protein with a 32 kDa peptide core. However, virions produced in HBE cultures, RSV (HBE), contain an even larger, 170 kDa, G protein (LgG). Here we show that LgG is found in virions from both subgroups A and B lab-adapted and clinical isolates. Unexpectedly, RSV (HBE) virions were approximately 100-fold more infectious for HBE cultures than for HEp-2 cells. Surprisingly, the cause of this differential infectivity, was reduced infectivity of RSV (HBE) on HEp-2 cells rather than enhanced infectivity on HBE cultures. The lower infectivity of RSV(HBE) for HEp-2 cells is caused by the reduced ability of LgG to interact with heparan sulfate proteoglycans (HSPG), the RSV receptor on HEp-2 cells. The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell.     

Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.     

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

Purification of human respiratory syncytial virus fusion glycoprotein

Human respiratory syncytial virus (RSV) fusion glycoprotein (F) elicits neutralizing antibodies to RSV and has therefore attracted much attention as a suitable candidate antigen in the development of gene-based vaccines against RSV infections. However, a major obstacle in vaccine development has been the problem of antigen purification. To address this problem, we have developed a new method that combines sucrose gradient ultracentrifugation and a two-step chromatographic process, to purify RSV F from RSV particles propagated in HEp-2 cells. Analysis of the fractions produced using this method showed recovery of a functional homodimer with a molecular weight of 140 kDa, and 54% preservation of the original F.     

Infection with respiratory syncytial virus enhances expression of native receptors for non-pilate Neisseria meningitidis on HEp-2 cells

Respiratory virus infections have been suggested to be predisposing factors for meningococcal disease. Respiratory syncytial virus (RSV) affects young children in the age range at greatest risk of disease caused by Neisseria meningitidis. It has been previously shown that glycoprotein G expressed on the surface of RSV-infected HEp-2 cells (a human epithelial cell line) contributed to higher levels of binding of meningococci compared with uninfected cells. The aim of the present study was to examine the effect of RSV infection on expression of surface molecules native to HEp-2 cells and their role in bacterial binding. Flow cytometry and fluorescence microscopy were used to assess bacterial binding and expression of host cell antigens. Some molecules analysed in this study have not been reported previously on epithelial cells. RSV infection significantly enhanced the expression of CD15 (P < 0.05), CD14 (P < 0.001) and CD18 (P < 0.01), and the latter two contributed to increased binding of meningococci to cells but not the Gram-positive Streptococcus pneumoniae.     

Antibody-dependent cell-mediated cytotoxicity against cells infected with respiratory syncytial virus: characterization of in vitro and in vivo properties

An in vitro 51Cr release assay for human antibody-dependent cell-mediated cytotoxicity (ADCC) against HeLa cells infected with respiratory syncytial virus (RSV) has been characterized by using leukophoresed and adherent cell-depleted adult lymphocytes. Lymphoytes from RSV seronegative children were also competent as effector cells. Sera from children with :1) primary and recurrent natural RSV infections, or 2) live attenuated RSV vaccine infection were examined to characterize the behavior of ADCC antibody in vivo. After natural RSV infection ADCC antibody rose and fell more rapidly than neutralizing antibody. In two children undergoing primary RSV infection with attenuated vaccine, neutralizing antibody was formed in the absence of detectable ADCC antibody. The nonparallel behavior of ADCC and neutralizing antibodies suggests the heterogeneity of either the antigen involved or the mechanism of antibody production in the two antibody systems.     

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.     

Current progress on development of respiratory syncytial virus vaccine

Human respiratory syncytial virus (HRSV) is a major cause of upper and lower respiratory tract illness in infants and young children worldwide. Despite its importance as a respiratory pathogen, there is currently no licensed vaccine for prophylaxis of HRSV infection. There are several hurdles complicating the development of a RSV vaccine: 1) incomplete immunity to natural RSV infection leading to frequent re-infection, 2) immature immune system and maternal antibodies of newborn infants who are the primary subject population, and 3) imbalanced Th2-biased immune responses to certain vaccine candidates leading to exacerbated pulmonary disease. After the failure of an initial trial featuring formalin-inactivated virus as a RSV vaccine, more careful and deliberate efforts have been made towards the development of safe and effective RSV vaccines without vaccine-enhanced disease. A wide array of RSV vaccine strategies is being developed, including live-attenuated viruses, protein subunit-based, and vector-based candidates. Though licensed vaccines remain to be developed, our great efforts will lead us to reach the goal of attaining safe and effective RSV vaccines in the near future.     

人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定

人呼吸道合胞病毒（human respiratory syncytial virus, RSV)基质蛋白（matrix protein,M）在RSV形态发生上具有重要作用,因含有CTL抗原表位,在疫苗研究上具有一定意义。为此,应用RT-PCR 方法从感染RSV的HEp-2 细胞中扩增获得M蛋白基因,构建了含M基因的非复制型重组腺病毒并进行表达和鉴定。基因序列分析显示RSV M基因仅有一处碱基发生错义突变。非复制型重组腺病毒DNA分子FGAd/RSVM转染293细胞,观察到细胞出现CPE,RT-PCR发现M基因有转录,Western blotting及间接免疫荧光分析检测到M蛋白。成功克隆A亚型RSV Long株M基因,并获得一株可表达A亚型RSV M蛋白的非复制型重组腺病毒FGAd/RSVM,可用于体内研究观察其免疫效果及免疫保护作用。     

Role for innate IFNs in determining respiratory syncytial virus immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1(-/-) mice that are incapable of generating Th1 CD4(+) cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-alphabetaR (IFN-alphabetaR(-/-)), the IFN-gammaR (IFN-gammaR(-/-)), or both receptors (IFN-alphabetagammaR(-/-)). Although both the IFN-gammaR(-/-) and the IFN-alphabetagammaR(-/-) animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8(+) T cells could be detected in lungs of IFN-alphabetagammaR(-/-) animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4(+) Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

Priming with live respiratory syncytial virus (RSV) prevents the enhanced pulmonary inflammatory response seen after RSV challenge in BALB/c mice immunized with formalin-inactivated RSV.

To investigate enhanced disease associated with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine, we studied the pulmonary inflammatory response to RSV in BALB/c mice immunized with live RSV, FI-RSV, or combinations of the two. After RSV challenge, the number of granular cells, the ratio of CD4+/CD8+ lymphocytes, and the level of Th2-like cytokine mRNAs in the bronchoalveolar lavage specimens in mice immunized first with live RSV and then with FI-RSV were lower than that in FI-RSV-immunized mice and close to that in live RSV-immunized mice. These data suggest that prior live RSV infection prevents most of the enhanced inflammatory response seen in FI-RSV-immunized mice and might explain lack of enhanced disease in older FI-RSV-immunized children. A live RSV vaccine might similarly decrease the risk of enhanced disease with non-live RSV vaccines.     

Phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and significant morbidity and mortality in the elderly, immunosuppressed, and immunocompromised patients and in patients with chronic lung diseases. Recently, we reported that the pulmonary surfactant phospholipid palmitoyl-oleoyl-phosphatidylglycerol (POPG) inhibited RSV infection in vitro and in vivo by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice markedly attenuated infection and associated inflammatory responses. Based on these findings, we expanded our studies to determine whether POPG is effective for prophylaxis and postinfection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2–1.0 mg/ml at 24 h after RSV infection of HEp-2 cells suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2–6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making postinfection application impractical. Intranasal administration of POPG (0.8–3.0 mg), 45 min before RSV inoculation in mice reduced viral infection by 1 log unit, suppressed inflammatory cell appearance in the lung, and suppressed virus-elicited interferon-γ production. These findings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection and that it has potential for application in humans.     

Improved effector activity and memory CD8 T cell development by IL-2 expression during experimental respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection in young children and the elderly. Studies of mice suggest that RSV suppresses the effector activity of CD8 T cells and the development of pulmonary CD8 T cell memory, in which the impaired effector activity could be recovered by in vitro IL-2 treatment. To investigate the effect of in vivo IL-2 expression on RSV immunity, mice were infected with RSV followed by administration of replication-defective adenovirus expressing IL-2. The effector activity of RSV M2-specific CD8 T cells and the development of CD8 T cell memory in the lung was significantly increased by IL-2 expression. Furthermore, the Ab responses against RSV were augmented by IL-2. Interestingly, weight loss and illness caused by RSV challenge were substantially reduced by IL-2 priming, suggesting that the pathogenesis of RSV-related disease could be prevented by IL-2-mediated enhancement of beneficial immune responses. Thus, our results show that IL-2 has potential to be used as a vaccine adjuvant against RSV infection.     

Recombinant respiratory syncytial virus lacking secreted glycoprotein G is attenuated, non-pathogenic but induces protective immunity

Respiratory syncytial virus (RSV) causes intense pulmonary inflammatory responses in some infected infants. The surface attachment protein 'G' of RSV has membrane-bound and secreted forms and shows homology to the CX3C chemokine fractalkine. Using recombinant techniques, we generated replication-competent recombinant clonal RSV expressing normal G proteins ('rRSV') or only the membrane-bound form of G ('Gmem rRSV'). Both recombinants grew well in HEp-2 cells, but after primary intranasal infection in mice, pulmonary Gmem rRSV replication was reduced tenfold compared to parental or rRSV; moreover, CCL2 and CCL5 production was greatly reduced and no apparent disease or pulmonary cellular infiltration was observed. However, Gmem rRSV-infected mice developed good antibody responses and were fully protected against subsequent intranasal challenge with parental virus. Even in mice sensitized to G by cutaneous infection with recombinant vaccinia expressing G, intranasal challenge with Gmem rRSV caused insignificant disease. We conclude that secreted G is a key viral product assisting virus replication in vivo, enhancing CCL2 and CCL5 production and promoting illness. Engineered RSV mutants lacking the ability to secrete G are thus promising vaccine candidates.     

CD8 T cells inhibit respiratory syncytial virus (RSV) vaccine-enhanced disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-gamma-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.     

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8<Superscript>+</Superscript> T-cell immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8⁺ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8⁺ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.     

稳定表达呼吸道合胞病毒非结构蛋白NS1细胞的构建及其生物特性研究

获得稳定表达RSV病毒NS1基因的HEp-2-NS1细胞株并对其生物学特性进行初步研究。采用RT-PCR方法从RSV病毒中获得NS1全长基因,克隆到逆转录病毒载体pBABE-puro中,重组载体与包装质粒PIK通过磷酸钙共沉淀法转染包装细胞293FT细胞,产生的逆转录病毒颗粒感染HEp-2细胞,经嘌呤霉素筛选后得到稳定表达细胞株,用QPCR、细胞病变染色法、RT-PCR和间接免疫荧光法检测细胞中NS1 mRNA和蛋白表达情况。结果表明重组逆转录病毒载体pBABE-NS1经双酶切及测序鉴定正确;筛选获得5株阳性单克隆细胞株,QPCR结果显示HEp-2-NS1细胞有NS1基因扩增,其中d株的相对表达量是正常细胞对照组的8 483倍;在外源干扰素作用下,HEp-2-NS1细胞仍对VSV病毒保持敏感,细胞感染病毒48h后染色结果显示全部死亡;对第3代及第30代细胞的RT-PCR和间接免疫荧光法检测结果显示,导入的NS1基因在HEp-2细胞中不仅能转录成相应的mRNA而且能成功稳定地表达。成功构建了稳定表达RSV病毒非结构蛋白NS1的HEp-2-NS1改造细胞株,为建立适用于临床分离的转基因细胞提供良好的基础。     

RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.