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Respiratory Syncytial Virus Immunobiology and Pathogenesis

Respiratory Syncytial Virus Immunobiology and Pathogenesis

297, 1–7 (2002) doi:10.1006/viro.2002.1431

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Respiratory Syncytial Immunobiology and Pathogenesis

Barney S. Graham,1 John A. Rutigliano, and Teresa R. Johnson

Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious , National Institutes of Health, Bethesda, Maryland 20892-3017 Received February 22, 2002; accepted February 21, 2002

Respiratory syncytial virus (RSV) is a pneumovirus in though underlying asthma and chronic bronchitis can be the family Paramyxoviridae. It is a pleomorphic envel- exacerbated during infection. However, in the institution- oped virus about 150 nm in diameter, transmitted by alized elderly, can be severe and in some stud- large particle aerosol and fomites, resulting in yearly ies has been associated with as much excess mortality epidemics ofrespiratory disease. RSV infectsnearly 70% as (Falsey et al., 1995). The pathology is less of infants in their first year of life and everyone by age distinctive in this setting and is often complicated by three (Glezen and Denny, 1973). Reinfection is common. chronic lung disease, heart failure, and bacterial super- About 50% of children infected in the first year of life are infection. Therefore, it is difficult to ascribe disease reinfected in the second year of life, and everyone is pathogenesis to a direct manifestation of virus-mediated reinfected every two to three years throughout life. cytopathology and virus-induced , or to ex- RSV disease syndromes. There are several distinct acerbation ofunderlying conditions. disease syndromes associated with RSV infection. While RSV infection is associated with high mortality in pa- most infants have mild disease, about 5% of those less tients with . This is particularly true in than one year ofage require hospitalization. The peak the setting ofsevere combined immunodeficiencydis- hospitalization rate is in infants between 2 and 3 months ease (Fishaut et al., 1980), and in patients following ofage. Those with bronchopulmonary dysplasia and allogenic bone marrow (Hertz et al., 1989) or lung trans- congenital heart disease are at greatest risk, but most plantation (Krinzman et al., 1998). Solid organ transplant hospitalized children have no apparent underlying con- recipients, patients with malignancies, or persons with dition. The virus infects both ciliated and nonciliated other selected immune deficiencies have milder disease. cells in small (75–300 ␮m in diameter) airways, resulting The disease syndrome is a giant cell pneumonia with in a bronchiolitis characterized clinically by wheezing. widespread syncytium formation in epithelial cells, pre- Bronchoalveolar lavage (BAL) reveals a predominance of sumably a direct result ofvirus-induced cytopathology. polymorphonuclear leukocytes in respiratory secretions. Finally, RSV has a legacy ofvaccine-enhanced dis- However, tissue sections reveal a predominance oflym- ease. Four studies using a Formalin-inactivated, alum- phocytes in the peribronchiolar and perivascular spaces, precipitated whole virus vaccine (FI-RSV) were per- and small airways containing fibrin, mucus, and a mix- formed in children in the mid-1960s. Vaccine-induced ture ofsloughed epithelial cells and inflammatory cells. immune responses did not protect against subsequent In addition, RSV infects the alveolar epithelium, particu- natural infection, and disease severity was increased. larly Type 1 alveolar pneumocytes. This component of Among vaccinated children in the youngest age group, RSV pathology is underappreciated despite signs of 80% required hospitalization and some died (Kim et al., lower airway disease such as hypoxia that are com- 1969). This tragedy has motivated extensive study of monly found even in patients. disease pathogenesis in animal models over the last 25 Most normal children and adults experience mild up- years and led to the current paradigms that guide vac- per respiratory disease during reinfection with RSV, al- cine development and approaches to therapy. Serological studies in the children with RSV vaccine- enhanced illness showed poor neutralizing activity and 1 To whom correspondence and reprint requests should be addressed fusion-inhibiting activity in the response to vac- at Building 40, Room 2502, 40 Convent Drive, MSC-3017, Bethesda, MD cine (Murphy et al., 1986, 1988), perhaps explaining the 20892-3017. Fax: (301) 480-2771. E-mail: [email protected]. lack of protection from natural infection. However, this

1 0042-6822/02 2 MINIREVIEW does not explain why illness was enhanced. They were sponses induced by RSV has distinct advantages be- also found to have high lymphoproliferative responses to cause ofthe wealth ofimmunological reagents and ge- RSV (Kim et al., 1976) and a relatively high netically defined strains available. frequency of eosinophilia (Chin et al., 1969). Tissue sec- The Th2 hypothesis of RSV pathogenesis. In mice tions from the children who died demonstrate a typical immunized with FI-RSV and then challenged with RSV, distribution ofinflammationaround the small airways, one sees an inflammatory infiltrate composed of mono- but the composition of the infiltrate is different from cytes, lymphocytes, and numerous eosinophils demon- primary infection with an abundance of eosinophils and strated by their large cytoplasmic granules (Fig. 2C). This neutrophils in addition to mononuclear cells (Fig. 1). is reminiscent ofthe pathology seen in infantswith There was no evidence ofextensive, virus-induced cyto- FI-RSV vaccine-enhanced disease. The pathology is de- pathology. These observations suggested a virus-spe- pendent on CD4ϩ T cells. Lung extracts from mice chal- cific cell-mediated immune process was responsible for lenged with RSV after FI-RSV immunization show a dom- the enhanced illness. inant IL-4 mRNA pattern, whereas the dominant re- The findings in animal models that relate to the syn- sponse in mice immunized with live RSV given drome ofthe RSV vaccine-enhanced illness will be re- intramuscularly or intranasally is IFN-␥ (Graham et al., viewed. In particular, how the current understanding of 1993). These findings suggested that an aberrant CD4ϩ vaccine-enhanced illness pathogenesis can inform our Th2 response may be the basis for the RSV vaccine- understanding ofdisease associated with primary RSV enhanced illness syndrome. The mechanism by which infection will be addressed. The central focus will be on this occurs is still controversial as discussed below. how prior exposure can induce aberrant adap- When the RSV vaccine-enhanced illness was associ- tive immune responses, and how those responses can ated with CD4ϩ Th2 responses, it promoted an interest in cause disease. The pathogenesis ofRSV in the institu- the role ofType 2 in the pathogenesis of tionalized elderly or immunocompromised patients will primary RSV infection. Primary RSV infection is associ- not be discussed in detail. ated with wheezing, and severe disease requiring hos- Murine model of RSV. A murine model ofRSV has been pitalization is associated with a higher incidence ofsub- used to study the pathogenesis ofvaccine-enhanced sequent childhood asthma (Sigurs et al., 1995). Some illness. Most work has been done in BALB/c mice that groups have shown RSV-specific IgE can be found in are relatively permissive to RSV replication compared to respiratory secretions ofchildren with severe disease other strains. However, the murine model has some (Welliver et al., 1981), and others have shown an increase limitations that should be recognized in the interpretation ofeosinophilic cationic protein (Garofalo et al., 1992), ofresults. The infectionis initiated in anesthetized mice findings consistent with Type 2 activity. How- by intranasal inoculation ofa large volume ofhigh titer ever, others have shown children with severe disease virus. The inoculum is aspirated directly into lung. Virus have a dominant IFN-␥ response without direct evidence replication occurs primarily in the alveolar parenchyma ofthe classical Type 2 cytokines IL-4 or IL-5 (Branden- in Type 1 pneumocytes (Figs. 2A and 2B). There is evi- burg et al., 2000). dence ofbronchiolar cytopathology in some strains, and How is the RSV-specific Th2 response induced in FI- immune infiltration occurs around arterioles and bron- RSV vaccine-enhanced illness? The RSV G glycoprotein chioles, as well as in the alveolar interstitium. Illness is is part ofthe envelope spike on the surfaceofthe virus mediated predominantly by the response to infec- and is the putative attachment protein. G has a number tion (Graham et al., 1991b). Therefore, the murine model ofinteresting structural features.It is a type II integral provides a good system to ask how the composition of membrane protein anchored at the amino terminus. the inflammatory infiltrate is regulated in response to About 50% ofthe protein produced is secreted fromthe RSV infection. It is not a good model for studying disease infected cell because of an alternative initiation codon in caused by virus-induced cytopathology in airway epithe- the transmembrane domain (Roberts et al., 1994). About lium. It is also not a good system for studying the kinetics 60% ofthe molecular weight is O-glycosylation, and there ofprimary RSV infectionor the role ofearly events me- is a high serine and threonine content and a very high diated by innate immunity, because the inoculum is so proline content making it more homologous to mucins high and is delivered directly into the lung. These types than any known viral glycoprotein. The glycosylation is ofquestions should be addressed in systems in which concentrated on either end ofthe molecule, and in the the virus is specific for the , and a low inoculum can central portion there is a cysteine noose with a known initiate infection in the upper respiratory tract that sub- heparan sulfate binding domain (Feldman et al., 1999) sequently spreads to the lower airway. Examples include and a CX3CR binding motif(Tripp et al., 2001). pneumonia virus ofmice (PVM) or Sendai infectionin RSV G has equally interesting antigenic properties. mice, BRSV in calves, or RSV in humans. With these Despite careful analysis, there has never been a G- caveats in mind, using the murine model to study the specific CD8ϩ T cell response reported. The reason for composition and regulation ofadaptive immune re- this is not known. It is probably not due to a defect in MINIREVIEW 3

FIG. 1. Haematoxylin and eosin-stained lung section fromthe autopsy ofa child who died in the one ofthe original FI-RSV trials complicated by vaccine-enhanced illness. The box in (A) framing a segment of the bronchiole is shown at higher magnification in (B). One of the many eosinophils is marked by a white arrow. FIG. 2. RSV infection of BALB/c mice is restricted primarily to the alveolar epithelial cells. (A) In situ hybridization ofRSV RNA in a lung section taken on day 4 after RSV challenge. (B) Transmission electron micrograph of Type 1 alveolar pneumocytes with budding filamentous RSV on day 4 of infection. The bar represents 200 nm. (C) Transmission electron micrograph of lung section from a mouse immunized with FI-RSV and subsequently challenged with RSV. Eosinophils are demonstrated by white arrows and by the presence oflarge cytoplasmic granules. FIG. 3. Impact ofCD8 ϩ CTL killing mechanisms on RSV pathogenesis. (A) illustrates the process ofclearing RSV infectionunder normal circumstances. CD8ϩ cytotoxic T lymphocytes (CTL) recognize RSV-infected epithelial cells through T cell receptor interactions with RSV-derived peptides presented in MHC class I molecules. The majority ofinfectedcells are eliminated via secretion ofperforinand granzymes by CTL. However, FasL expression (lightning bolt) by activated CTL also participates in mediating ofRSV-infectedcells. In addition, Type 1 cytokines, su ch as IFN-␥ and TNF-␣, are secreted by CTL in modest amounts. These combined processes are hypothesized to selectively induce apoptosis (Red X) in RSV-infected cells with very little injury of uninfected cells. (B) represents the immune response hypothesized to occur in the setting of Type 2 cytokines, particularly IL-4. Increased IL-4 leads to upregulation ofFasL on activated CTL. Since most cells express a basal level ofFas, the augmented expression ofFasL on CTL may result in bystander killing ofuninfectedcells along with killing ofRSV-infectedcells. In addition, the less efficient CTL produce exaggerated levels of IFN-␥ and TNF-␣. The altered CTL activity in this setting is hypothesized to increase bystander killing and to cause more severe lung pathology and enhanced illness. 4 MINIREVIEW processing MHC class I-restricted epitopes contained able in the extracellular milieu for processing through the within G, since the H-2Kd-restricted CTL epitope from M2 endocytic pathway and presentation to CD4ϩ T cells. inserted into G and expressed in vaccinia can induce a This may allow certain G-specific CD4ϩ T cell responses CTL response (Srikiatkachorn and Braciale, 1997). The to occur prior to the modulating influences of IFN-␥ lack ofMHC class I-restricted response to G suggests produced by CD8ϩ T cells and NK cells. In addition, the that the memory T cell response to G is heavily biased secreted G can bind to heparan sulfate moieties (Feld- toward MHC class II-restricted CD4ϩ T cells. Openshaw man et al., 1999), fractalkine receptor (Tripp et al., 2001), et al. (1992) first showed that mice immunized with re- or lectin-like proteins (Barr et al., 2000), perhaps trigger- combinant vaccinia expressing RSV G develop an eosin- ing signaling events that influence subsequent immune ophil response in lung and BAL following RSV challenge. responses. Recombinant vaccinia expressing WT G, but none ofthe We have asked whether the property ofG secretion is other RSV proteins, results in a significant percentage of important for inducing the Type 2 cytokine profile, eosin- eosinophils in BAL. They subsequently showed this re- ophilia, and pathology associated with the vaccine-en- sponse was dependent on the genetic background ofthe hanced illness. Recombinant vaccinia encoding mouse (Hussell et al., 1998). Several groups then WT G (vvGwt), membrane-anchored G (vvGr), or secreted showed that G contains a dominant MHC class II-re- G (vvGs) were produced by Sharon Roberts in the labo- stricted epitope (aa 184–198) that induces both Th1 and ratory ofDr. Gail Wertz (Roberts et al., 1994). The vaccinia Th2 CD4ϩ T cells (Tebbey et al., 1998; Sparer et al., 1998; constructs were inoculated by intradermal injection at Srikiatkhachorn et al., 1999). This epitope is in the region the base ofthe tail, and RSV challenge was performed6 ofthe heparan sulfatebinding domain and just down- weeks later. We first asked how immunization affected stream ofthe cysteine noose structure. Varga et al. then eosinophil responses in BAL following subsequent RSV showed the CD4ϩ T cells responding to this epitope infection. Mock immunization or vacF does not lead to predominantly utilize V␤14 in their T cell receptors (Varga postchallenge eosinophil responses. In contrast FI-RSV et al., 2001). and vvGs induce a robust eosinophilia. Membrane-an- These unique properties ofG begged the question, chored G induces eosinophilia, but to a much lesser were G-specific immune responses responsible for the extent, and the vvGwt (a mixture ofboth membrane RSV vaccine-enhanced illness? This has obvious impli- anchored and secreted G) induces an intermediate re- cations for vaccine development since if true, G deter- sponse (Johnson et al., 1998). We next asked whether the minants could simply be removed from vaccine products. eosinophil response required IL-4, using both antibody However, most ofthe antigenic variation present in RSV inhibition with the anti-IL-4 monoclonal antibody, 11B.11, isolates occurs in the carboxyl-terminus ofG, suggesting and IL-4 knockout mice (Johnson et al., 1999). Earlier that G is an important target for the immune response studies had shown that FI-RSV-induced eosinophilia fol- and is under selective pressure to change. Therefore, lowing subsequent challenge was IL-4 dependent (Tang including G in vaccine formulations would be advanta- et al., 1994). These studies showed that the eosinophilia geous. induced by RSV infection following immunization with the There are several arguments that G antigenic deter- secreted G does not require IL-4. This led us to ask minants were not the essential factor in the FI-RSV vac- whether the G-induced response was associated with cine-enhanced illness. They include the following: IL-13, which is induced at high levels after G immuniza- tion. We used IL-4 knockout mice in combination with an 1. G is expressed on wild-type virus, which typically IL-13 receptor antagonist. As before we showed that the leads to a Th1-type response in primary infection. G-induced eosinophilia occurred in IL-4 KO mice, 2. The G-induced response in mice is dependent on whereas the FI-RSV induced eosinophilia does not occur the genetic background, is epitope dependent, and is in IL-4 KO mice. Similarly, IL-13 receptor antagonist alone restricted to selected T cell receptors utilizing V␤14. In did not attenuate G-induced eosinophilia, but did elimi- contrast, FI-RSV immunization seemed to lead to en- nate FI-RSV-induced eosinophilia. Interestingly, ifIL-13 hanced disease in virtually all individuals, suggesting receptor antagonist is given to IL-4 KO mice, G is not that it was not dependent on the genetic background or able to induce eosinophils. This indicates that IL-4 and restricted to a single epitope or T cell receptor. IL-13 are able to compensate for each other to allow 3. Pathology, illness, and immune responses similar G-induced eosinophilia, but both cytokines are needed to those elicited by FI-RSV can be elicited by purified RSV for FI-RSV to induce the response (T. R. Johnson et al., F glycoprotein, especially when formulated in alum (Gra- submitted). ham et al., 1993; Hancock et al., 1996). These findings support the concept that G-specific However, there are other properties ofG that could epitopes were not necessarily responsible for eliciting affect the virus-specific immune response. G is produced the vaccine-enhanced illness and suggest that the pat- early in the infected cell and secreted (Hendricks et al., tern ofimmunizing parenterally with an antigen pro- 1987). Therefore it is one of the first RSV proteins avail- cessed and presented through the endocytic pathway in MINIREVIEW 5

MHC class II molecules followed by airway infection with sensitization with ovalbumin (Peebles et al., 2001a) or RSV has the potential to elicit a Type 2 immune re- priming with pertussis toxin (Samore and Siber, 1996; sponse. This sequence ofparenteral immunization fol- Fischer et al., 1999). In a murine model ofPVM eosino- lowed by airway challenge is known to elicit airway philia is seen following primary infection, but it is not a hypersensitivity and eosinophil responses even when consequence ofIL-5 and the adaptive immune response. using a model antigen such as ovalbumin (Willis-Karp, Rather, it occurs early in infection and is mediated largely 1999). As mentioned above, purified F formulated in alum by MIP-1␣ and other components ofthe innate immune followed by RSV infection also results in airway eosino- response (Domachowske et al., 2000). This model ofa philia (Graham et al., 1993). Likewise, priming mice with pneumovirus matched to its natural host may better re- recombinant vaccinia expressing a secreted F glycopro- flect the early events following infection than the model tein, as opposed to the wild-type membrane-anchored F, using human RSV in the mouse. It is possible that in also induces IL-4 production following RSV challenge children with underlying atopy or other factors predis- (Bembridge et al., 1999). Therefore, any immunization posing to a Type 2 cytokine environment the response to strategy in RSV-naı¨ve infants based on products with primary RSV infection may be more likely to involve Th2 obligate processing through the endocytic pathway and CD4ϩ T cells. This has been demonstrated in mice presentation strictly to CD4ϩ T lymphocytes may have (Peebles et al., 2001a), but more work is needed in clinical the capacity to elicit the vaccine-enhanced illness. studies to define whether this happens in humans. RSV has other properties that may promote Type 2 How do Type 2 cytokines influence disease severity cytokine production. RSV shares a legacy ofvaccine- during virus infection? While there are many observa- enhanced illness with measles. In both cases vaccina- tions ofpathogens that have more severe disease in the tion was with whole inactivated virus that elicited poor setting ofexcess Type 2 cytokine production, the mech- functional antibody responses. There is evidence in both anisms ofdisease enhancement are poorly defined.In cases that the enhanced illness was caused by an ab- the airway, increased mucus production could contribute errant T cell response. Both viruses are in the family to airway obstruction, or eosinophil recruitment and de- Paramyxoviridae and share several properties. In partic- granulation could lead to direct tissue damage. Th2 ular, both viruses have innate mechanisms for inhibiting CD4ϩ T cells are not inherently more pathogenic, and in the effects of IFN-␣/␤ (Naniche et al., 2000; Schlender et the setting ofautoimmunity they are less destructive than al., 2000). The NS1 and NS2 proteins ofbovine RSV have Th1 CD4ϩ T cells. In many cases it appears that the been shown to antagonize the antiviral effects of IFN-␣/␤ mechanisms for clearance of the are less (Schlender et al., 2000). Durbin et al. have also shown effective, resulting in direct tissue damage from the that the lack ofIFN- ␣/␤-mediated signaling during RSV pathogen itself. However, in the murine model of RSV infection promotes eosinophilia and Type 2 cytokine pro- and other viruses, the enhanced illness is not related to duction (Durbin et al., 2002). It seems likely that this may direct pathogen-induced cytopathology, but is dependent be an important determinant in why RSV is associated on the immune response. Therefore, we asked how IL-4 with vaccine-enhanced illness and other disease syn- influenced the mechanisms of RSV clearance and how dromes associated with Type 2 cytokine production. these immunological effectors influenced disease patho- However, more work is needed on how IFN-␣/␤ influ- genesis. Two early observations led us to focus on CD8ϩ ences the differentiation of T cells, and how interference T cells as a key mediator ofthis process. First, inhibiting with these pathways contributes to the pathogenesis of IL-4 during FI-RSV immunization improved CD8ϩ T cell RSV and other virus infections. response following subsequent challenge and led to Do Type 2 cytokines play a role in the pathogenesis of reduced illness (Tang and Graham, 1994). Second, CD8ϩ severe primary RSV infection? RSV infections in young T cell-mediated RSV clearance is diminished and pathol- infants requiring hospitalization are associated with ev- ogy is enhanced in IL-4 overexpressing mice (Fischer et idence ofeosinophil cationic protein in airway secre- al., 1997). These findings suggested that IL-4 may be tions. However, it is not clear whether disease in this altering functional properties of CD8ϩ T cells, causing setting is a manifestation of Type 2 cytokine production them to have less efficient cytolytic function and to pro- or not. Type 2 cytokines are not found in respiratory duce more pathology. secretions ofthese infants.In contrast, large amounts of We therefore constructed recombinant vaccinia vi- IFN-␥ are found (Brandenburg et al., 2000). It is possible ruses to express the RSV M2 protein that contains a that Type 2 cytokines are acting locally, but evaluation of defined H-2Kd-restricted T cell epitope with or without cytokine production from peripheral blood mononuclear coexpression ofIL-4. Using these vectors, we showed cells (PBMCs) ofRSV-infectedchildren (Anderson et al., that IL-4 coexpression reduced the total cytolytic capac- 1994) and studies in animal models do not bear that out. ity ofthe M2-specificCD8 ϩ T cells (Aung et al., 1999). Primary infection with RSV in mice does not induce This raised the question ofwhich cytolytic mechanisms significant levels of Type 2 cytokine production unless were affected. Naı¨ve CD8ϩ T cells recognize processed there is some prior stimulus such as allergic airway peptides in the context ofMHC class I molecules and 6 MINIREVIEW become activated after costimulation through interaction vaccinia virus expressing respiratory syncytial virus M2 protein. between B7 and CD28. This leads to upregulated expres- J. Virol. 73, 8944–8949. Barr, F. E., Pedigo, H., Johnson, T. R., and Shepherd, V. L. (2000). sion ofselected molecules. When a virus-infectedcell is Surfactant protein-A enhances uptake of respiratory syncytial virus detected by TCR interaction with the relevant epitope, by monocytes and U937 macrophages. Am. J. Respir. Cell Mol. Biol. perforin is released and allows the entry of granzymes 23, 586–592. and serine proteases into the target cell. This is a cal- Bembridge, G. P., Lopez, J. A., Bustos, R., Melero, J. A., Cook, R., Mason, cium-dependent process. In addition, FasL expression is H., and Taylor, G. (1999). Priming with a secreted form of the fusion protein ofrespiratory syncytial virus (RSV) promotes interleukin-4 upregulated and interaction with Fas can lead to cal- (IL-4) and IL-5 production but not pulmonary eosinophilia following cium-independent signaling. Both pathways can lead to RSV challenge. J. Virol. 73, 10086–10094. apoptosis and cell death. In general perforin-mediated Brandenburg, A. H., Kleinjan, A., van Het Land, B., Moll, H. A., Timmer- lysis is more targeted and FasL-mediated lysis is less man, H. H., de Swart, R. L., Neijens, H. J., Fokkens, W., and Osterhaus, specific and more likely to result in antigen-independent A. D. (2000). Type 1-like immune response is found in children with respiratory syncytial virus infection regardless of clinical severity. bystander killing. J. Med. Virol. 62, 267–277. We found that coexpression of IL-4 with RSV M2 in Chin, J., Magoffin, R. L., Shearer, L. A., Schieble, J. H., and Lennette, recombinant vaccinia increases FasL expression on the E. H. (1969). Field evaluation ofa respiratory syncytial virus vaccine M2-specific CD8ϩ T cells resulting in diminished per- and a trivalent parainfluenza virus vaccine in a pediatric population. Am. J. Epidemiol. 89, 449–463. forin-mediated cytolytic activity and enhanced FasL-me- Domachowske, J. B., Bonville, C. A., Dyer, K. D., Easton, A. J., and diated target cell killing (Aung and Graham, 2000). Ex- Rosenberg, H. F. (2000). Pulmonary eosinophilia and production of periments in perforin KO mice confirmed that perforin MIP-1alpha are prominent responses to infection with pneumonia was not necessary for RSV clearance, but when other virus ofmice. Cell Immunol. 200, 98–104. cytolytic mechanisms are used, clearance and disease Durbin, J., Johnson, T. R., Durbin, R. K., Mertz, S., Peebles, R. S., Jr., and Graham, B. S. (2002). The role ofinterferonin RSV pathogenesis. expression are delayed, and illness is more prolonged. J. Immunol. 168, 2944–2951. In addition, excess production ofIFN- ␥ and TNF-␣ in Falsey, A. R., Cunningham, C. K., Barker, W. H., Kouides, R. W., Yuen, lung temporally correlated with the illness (Aung et al., J. B., Menegus, M., Weiner, L. B., Bonville, C. 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