Prevention and treatment of respiratory syncytial virus bronchiolitis and postbronchiolitic wheezing

Respiratory syncytial virus (RSV) is the primary cause of hospitalization for acute respiratory tract illness in general and specifically for bronchiolitis in young children. The link between RSV bronchiolitis and reactive airway disease is not completely understood, even though RSV bronchiolitis is frequently followed by recurrent episodes of wheezing. Therapy with ribavirin does not appear to significantly reduce long-term respiratory outcome of RSV lower respiratory tract infection, and corticosteroid or bronchodilator therapy may possibly improve outcomes only on a short-term basis. No vaccine against RSV is yet available. It is not known whether prophylaxis with RSV intravenous immune globulin or palivizumab can reduce postbronchiolitic wheezing.     

An epidemiological study of respiratory syncytial virus associated hospitalizations in Denmark

Respiratory syncytial virus (RSV) is the primary cause of hospitalization for acute respiratory tract illness in general and specifically for bronchiolitis in young children. The link between RSV bronchiolitis and reactive airway disease is not completely understood, even though RSV bronchiolitis is frequently followed by recurrent episodes of wheezing. Therapy with ribavirin does not appear to significantly reduce long-term respiratory outcome of RSV lower respiratory tract infection, and corticosteroid or bronchodilator therapy may possibly improve outcomes only on a short-term basis. No vaccine against RSV is yet available. It is not known whether prophylaxis with RSV intravenous immune globulin or palivizumab can reduce postbronchiolitic wheezing.     

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.     

IL-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4(+) and CD8(+) T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10(-/-) mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10(-/-) mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection.     

TARGETED THERAPY OF RESPIRATORY SYNCYTIAL VIRUS BY 2-5A ANTISENSE

Respiratory syncytial virus is a leading cause of respiratory disease in infants, young children, immunocompromized patients, and the elderly. Previous work has shown that RNase L, an antiviral enzyme of the interferon system, can be recruited to cleave RSV genomic RNA by attaching tetrameric 2′-5′-linked oligoadenylates (2 5A) to an antisense oligonucleotide complementary to repetitive intergenic sequences within the RSV genome (2 5A antisense). RBI034, a 2′-O-methyl RNA-modified analogue of the 2 5A anti-RSV compound, was found to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L· and sequence-specific manner. RBI034′s efficacy in suppressing RSV replication in cell culture is 50 to 100 times better than ribavirin, the only approved drug for RSV infection. Here we show that the activity of 2 5A antisense compound can be further enhanced by a combination treatment with interferon or ribavirin. The anti-RSV activity resulting from combination treatment is more potent than either treatment alone. We also demonstrate that RBI034 is effective against RSV in three different species: mice, cotton rats, and African green monkeys.     

Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats.

Initial studies of the prophylactic effect of parenterally administered respiratory syncytial virus (RSV)-neutralizing antibodies in cotton rats indicated that virus replication in lung tissues was restricted when animals with preexisting antibody titers in serum of 1:100 or more (as measured by plaque reduction) were challenged intranasally with 10(4) PFU of virus. Subsequently, a therapeutic effect of parenterally administered RSV antibodies (present in human gamma globulin) was demonstrated in both cotton rats and owl monkeys. Parenteral inoculation of RSV-infected cotton rats or owl monkeys with purified human immunoglobulin licensed for intravenous administration in humans (IVIG) effected a 10(-1.7) to 10(-2.7) reduction in the level of pulmonary virus at the height of infection. Because of these encouraging results, we examined topical administration of IVIG to determine whether it was also effective and whether it offered an advantage over the parenteral route with regard to simplicity and the dose required for full therapeutic effect. IVIG (0.025 g/kg) administered topically by the intranasal route to anesthetized cotton rats at the height of RSV infection effected a 10(2.2)-fold reduction in viral titers of pulmonary tissues and a complete clearance of detectable virus in 92% of the animals within 24 h. In contrast, 4 g of IVIG per kg was required to produce a comparable therapeutic effect when the material was administered parenterally. Thus, the therapeutic effect of IVIG was 160 times greater by the topical route than by parenteral inoculation.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

Respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the most common worldwide cause of lower respiratory tract infections (LRI) in infants less than 6 months of age. The prophylaxis against RSV infection by vaccination has been unsuccessful because of its adverse effects. As antiviral drug, ribavirin spray (aerosol) had been used clinically and reduces the amount of virus load, without reducing the necessity of symptomatic therapy and the duration of hospitalization. Therefore RSV LRI has been treated mainly symptomatically. Recently humanized anti-RSV F protein monoclonal antibody was developed and prescribed for prevention in high-risk infants such as premature ones and those with chronic lung and congenital heart diseases. It reduced the incidence of hospitalization significantly. It has been introduced in clinical use in Japan following to Western countries. On the other hand, a number of anti-RSV drugs have now been investigation; however, no valuable drugs for clinical use have been yet developed.     

Targeted therapy of respiratory syncytial virus by 2-5A antisense

Respiratory syncytial virus is a leading cause of respiratory disease in infants, young children, immunocompromized patients, and the elderly. Previous work has shown that RNase L, an antiviral enzyme of the interferon system, can be recruited to cleave RSVgenomic RNA by attaching tetrameric 2' 5'-linked oligoadenylates (2 5A) to an antisense oligonucleotide complementary to repetitive intergenic sequences within the RSV genome (2 5A antisense). RBI034, a 2'-O-methyl RNA-modified analogue of the 2 5A anti-RSV compound, was found to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. RBI034s efficacy in suppressing RSV replication in cell culture is 50 to 100 times better than ribavirin, the only approved drug for RSV infection. Here we show that the activity of 2 SA antisense compound can be further enhanced by a combination treatment with interferon or ribavirin. The anti-RSV activity resulting from combination treatment is more potent than either treatment alone. We also demonstrate that RBI034 is effective against RSV in three different species: mice, cotton rats, and African green monkeys.     

五种主要上呼吸道病毒多重RT-PCR 检测方法的建立

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。     

Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10.

In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.     

Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.     

Respiratory syncytial virus infection in C57BL/6 mice: clearance of virus from the lungs with virus-specific cytotoxic T cells.

We describe respiratory syncytial virus (RSV)-specific cytotoxic T-cell (CTL) lines and clones developed from the spleens of C57BL/6 and BALB/c mice. Line 7 and clones derived from it were H-2Kb restricted, whereas line 12 had both Kb and Db components. Both lines, and all the clones except one, could lyse targets infected with either strain A or strain B RSV. Line 7 or 7-11E1 cells (8 x 10(6) to 10 x 10(6) given intravenously cleared RSV from the lungs of infected mice. There was no morbidity or mortality in any of the infected mice whether or not they received T cells. The C57BL/6 mouse is a useful model system in which to study the role of the CTL response in protective immunity to RSV. CTL lines and clones can mediate clearance of RSV from the lungs of normal mice without producing any associated morbidity.     

Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10-producing cells in the lung during acute RSV infection were CD4(+) T cells. The IL-10-producing CD4(+) T cells included Foxp3(+) regulatory T cells, Foxp3(-) CD4(+) T cells that coproduce IFN-γ, and Foxp3(-) CD4(+) T cells that do not coproduce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8(+) and CD4(+) T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4(+) T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4(+) T cells and a concomitant decrease in Foxp3(+) regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.     

The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 x 10(2) PFU/gram tissue (C3H mice) to greater than 10(5) PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 x 10(5) PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.     

Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus

Respiratory syncytial virus (RSV) infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that certain polymorphisms in the gene encoding the regulatory cytokine IL-10 are associated with the development of severe bronchiolitis in RSV infected infants. Here, we examined the role of IL-10 in a murine model of primary RSV infection and found that high levels of IL-10 are produced in the respiratory tract by anti-viral effector T cells at the onset of the adaptive immune response. We demonstrated that the function of the effector T cell -derived IL-10 in vivo is to limit the excess pulmonary inflammation and thereby to maintain critical lung function. We further identify a novel mechanism by which effector T cell-derived IL-10 controls excess inflammation by feedback inhibition through engagement of the IL-10 receptor on the antiviral effector T cells. Our findings suggest a potentially critical role of effector T cell-derived IL-10 in controlling disease severity in clinical RSV infection.     

Interleukin-27 inhibits vaccine-enhanced pulmonary disease following respiratory syncytial virus infection by regulating cellular memory responses

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in young children. In the 1960s, infants vaccinated with formalin-inactivated RSV developed a more severe disease characterized by excessive inflammatory immunopathology in lungs upon natural RSV infection. The fear of causing the vaccine-enhanced disease (VED) is an important obstacle for development of safe and effective RSV vaccines. The recombinant vaccine candidate G1F/M2 immunization also led to VED. It has been proved that cellular memory induced by RSV vaccines contributed to VED. Interleukin-27 (IL-27) and IL-23 regulate Th1, Th17, and/or Th2 cellular immune responses. In this study, mice coimmunized with pcDNA3-IL-27 and G1F/M2 were fully protected and, importantly, did not develop vaccine-enhanced inflammatory responses and immunopathology in lungs after RSV challenge, which was correlated with moderate Th1-, suppressed Th2-, and Th17-like memory responses activated by RSV. In contrast, G1F/M2- or pcDNA3-IL-23+G1F/M2-immunized mice, in which robust Th2- and Th17-like memory responses were induced, developed enhanced pulmonary inflammation and severe immunopathology. Mice coimmunized with G1F/M2 and the two cytokine plasmids exhibited mild inflammatory responses as well as remarkable Th1-, suppressed Th2-, and Th17-like memory responses. These results suggested that Th1-, Th2-, and Th17-like memory responses and, in particular, excessive Th2- and Th17-like memory responses were closely associated with VED; IL-27 may inhibit VED following respiratory syncytial virus infection by regulating cellular memory responses.