Overexpression of interleukin-4 delays virus clearance in mice infected with respiratory syncytial virus.

Although interleukin-4 (IL-4) expression has been implicated in vaccine-enhanced respiratory syncytial virus (RSV) disease, its role in mediating the immune response to primary RSV infection remains unclear. To assess the effect of IL-4 production on typical RSV infection, transgenic mice which either overexpress or fail to express IL-4 were challenged intranasally with RSV and their responses were compared to those of the parent strains. IL-4-deficient mice eliminated virus from the lung as quickly as did C57BL/6 controls. In contrast, mice which constitutively overexpress IL-4 showed delayed virus clearance compared with mice of the FVB/N control strain, although peak viral titers did not differ. IL-4 overexpression increased the magnitude of the subsequent antibody response. Lung lymphocytes harvested from IL-4-overexpressing mice post-RSV challenge showed diminished RSV-specific cytolytic activity compared with controls. Both IL-4-deficient and IL-4-overexpressing strains resisted rechallenge. These data imply that constitutive IL-4 expression delays or suppresses the development of a virus-specific cytotoxic lymphocyte population important in clearing primary RSV infection.     

Secreted Respiratory Syncytial Virus G Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism

The attachment glycoprotein G of respiratory syncytial virus (RSV) is produced as both membrane-anchored and secreted forms by infected cells. Immunization with secreted RSV G (Gs) or formalin-inactivated alumprecipitated RSV (FI-RSV) predisposes mice to immune responses involving a Th2 cell phenotype which results in more severe illness and pathology, decreased viral clearance, and increased pulmonary eosinophilia upon subsequent RSV challenge. These responses are associated with increased interleukin-4 (IL-4) production in FI-RSV-primed mice, and the responses are IL-4 dependent. RNase protection assays demonstrated that similar levels of IL-4 mRNA were induced after RSV challenge in mice primed with vaccinia virus expressing Gs (vvGs) or a construct expressing only membrane-anchored G (vvGr). However, upon RSV challenge, vvGs-primed mice produced significantly greater levels of IL-5 and IL-13 mRNA and protein than vvGr-primed mice. Administration of neutralizing anti-IL-4 antibody 11.B11 during vaccinia virus priming did not alter the levels of vvGs-induced IL-5, IL-13, pulmonary eosinophilia, illness, or RSV titers upon RSV challenge, although immunoglobulin G (IgG) isotype profiles revealed that more IgG2a was produced. vvGs-priming of IL-4-deficient mice demonstrated that G-induced airway eosinophilia was not dependent on IL-4. In contrast, airway eosinophilia induced by FI-RSV priming was significantly reduced in IL-4-deficient mice. Thus we conclude that, in contrast to FI-RSV, the secreted form of RSV G can directly induce IL-5 and IL-13, producing pulmonary eosinophilia and enhanced illness in RSV-challenged mice by an IL-4-independent mechanism.     

IL-13 is required for eosinophil entry into the lung during respiratory syncytial virus vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.     

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.     

IL-13 is associated with reduced illness and replication in primary respiratory syncytial virus infection in the mouse

The role of IL-13 in respiratory syncytial virus (RSV) immunopathogenesis is incompletely described. To assess the effect of IL-13 on primary RSV infection, transgenic mice which either overexpress IL-13 in the lung (IL-13 OE) or non-transgenic littermates (IL-13 NT) were challenged intranasally with RSV. IL-13 OE mice had significantly decreased peak viral titers four days after infection compared to non-transgenic littermates. In addition, IL-13 OE mice had significantly lower RSV-induced weight loss and reduced lung IFN-gamma protein expression compared with IL-13 NT mice. In contrast, primary RSV challenge of IL-13 deficient mice resulted in a small, but statistically significant increase in viral titers on day four after infection, no difference in RSV-induced weight loss compared to wild type mice, and augmented IFN-gamma production on day 6 after infection. In STAT1-deficient (STAT1 KO) mice, where primary RSV challenge produced high levels of IL-13 production in the lungs, treatment with an IL-13 neutralizing protein resulted in greater peak viral titers both four and six days after RSV and greater RSV-induced weight loss compared to mice treated with a control protein. These results suggest that IL-13 modulates illness from RSV-infection.     

IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-gamma, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-gamma in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-gamma were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-gamma, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-gamma-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-gamma and IL-4 are not essential for these responses to RSV infection.     

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.     

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.     

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.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

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.     

Priming with a secreted form of the fusion protein of respiratory syncytial virus (RSV) promotes interleukin-4 (IL-4) and IL-5 production but not pulmonary eosinophilia following RSV challenge

The attachment (G) protein of respiratory syncytial virus (RSV) is synthesized as two mature forms: a membrane-anchored form and a smaller secreted form. BALB/c mice scarified with vaccinia virus (VV) expressing the secreted form develop a greater pulmonary eosinophilic influx following RSV challenge than do mice scarified with VV expressing the membrane-anchored form. To determine if a soluble form of an RSV protein was sufficient to induce eosinophilia following RSV challenge, a cDNA that encoded a secreted form of the fusion (F) protein of RSV was constructed and expressed in VV (VV-Ftm(-)). Splenocytes and lung lymphocytes from mice primed with VV-Ftm(-) produced significantly more of the Th2 cytokines interleukin-4 (IL-4) and IL-5 than did mice vaccinated with VV expressing either the native (membrane-anchored) form of the F protein or the G protein. Although mice scarified with VV-Ftm(-) developed a slight increase in the number of pulmonary eosinophils following RSV infection, the increase was not as great as that seen in VV-G-primed mice. Despite the increased IL-4 and IL-5 production and in contrast to mice primed with VV-G, mice primed with VV-Ftm(-) developed RSV-specific cytotoxic T lymphocytes (CTL) and maintained high levels of gamma interferon production. These data demonstrate that recombinant VV strains expressing soluble forms of RSV proteins induce immune responses that are more Th2-like. However, this change alone does not appear sufficient to induce vaccine-augmented disease in the face of active CD8(+) CTL populations.     

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.     

Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection.

Mice sensitized to the G (attachment) or F (fusion) glycoproteins of respiratory syncytial virus (RSV) expressed different patterns of cytokine production and lung pathology when challenged by intranasal infection with RSV. Five days after challenge, mice sensitized to G glycoprotein produced high levels of interleukin-4 (IL-4) and IL-5 in the lungs and spleens and developed extensive pulmonary eosinophilia, while mice sensitized to F glycoprotein produced IL-2 and developed a mononuclear cell infiltration. Memory lymphocytes isolated 2 weeks after intranasal challenge of mice primed to the G or F glycoprotein secreted only IL-2 and gamma interferon (IFN-gamma) when stimulated with RSV. IL-4 and IL-5 production characteristic of Th2-type effectors in the lung was observed only after multiple rounds of in vitro stimulation of RSV G-specific memory T lymphocytes with antigen. Also IFN-gamma production appeared to play only a minor role in the expression of pulmonary pathology characteristic of Th1 or Th2 T-lymphocyte responses, because mice genetically deficient in IFN-gamma production by gene disruption displayed the same pattern of pulmonary inflammation to RSV infection after priming to RSV F or G as conventional mice. These results suggest that effector T lymphocytes exhibit a different pattern of cytokine production than memory T-lymphocyte precursors precommitted to a Th1 or Th2 pattern of differentiation. Furthermore, these observations raise the possibility that the cytokine response of human memory T lymphocytes after a single exposure to antigen in vitro may not accurately reflect the cytokine response of differentiated effector T lymphocytes at the site of infection in vivo.     

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.     

The enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and IL-13 production

Respiratory syncytial virus (RSV) infection in early life is suspected to play a role in the development of post-bronchiolitis wheezing and asthma. Reinfection is common at all ages, but factors that determine the development of altered airway function after reinfection are not well understood. This study was conducted in a mouse model to define the role of age in determining the consequences on airway function after reinfection. Mice were infected shortly after birth or at weaning and were reinfected 5 wk later, followed by assessment of airway function, airway inflammation, and lung histopathology. Infection of mice at weaning elicited a protective airway response upon reinfection. In this age group, reinfection resulted in increased airway inflammation, but without development of airway hyperresponsiveness (AHR) or eosinophilia and decreased IL-13 levels. By contrast, neonatal infection failed to protect the airways and resulted in enhanced AHR after reinfection. This secondary response was associated with the development of airway eosinophilia, increased IL-13 levels, and mucus hyperproduction. Both CD4- and CD8-positive T cells were a source of IL-13 in the lung, and inhibition of IL-13 abolished AHR and mucus production in these mice. Inoculation of UV-inactivated virus failed to elicit these divergent responses to reinfection, emphasizing the requirement for active lung infection during initial exposure. Thus, neonatal RSV infection predisposes to the development of airway eosinophilia and enhanced AHR via an IL-13-dependent mechanism during reinfection, whereas infection at a later age protects against the development of these altered airway responses after reinfection.     

Virally delivered cytokines alter the immune response to future lung infections

Respiratory syncytial virus (RSV) is an important cause of infant morbidity and mortality worldwide and is increasingly recognized to have a role in the development and exacerbation of chronic lung diseases. There is no effective vaccine, and we reasoned that it might be possible to skew the immune system towards beneficial nonpathogenic responses by selectively priming protective T-cell subsets. We therefore tested recombinant RSV (rRSV) candidates expressing prototypic murine Th1 (gamma interferon [IFN-γ]) or Th2 (interleukin-4 [IL-4]) cytokines, with detailed monitoring of responses to subsequent infections with RSV or (as a control) influenza A virus. Although priming with either recombinant vector reduced viral load during RSV challenge, enhanced weight loss and enhanced pulmonary influx of RSV-specific CD8⁺ T cells were observed after challenge in mice primed with rRSV/IFN-γ. By contrast, rRSV/IL-4-primed mice were protected against weight loss during secondary challenge but showed airway eosinophilia. When rRSV/IL-4-primed mice were challenged with influenza virus, weight loss was attenuated but was again accompanied by marked airway eosinophilia. Thus, immunization directed toward enhancement of Th1 responses reduces viral load but is not necessarily protective against disease. Counter to expectation, Th2-biased responses were more beneficial but also influenced the pathological effects of heterologous viral challenge.     

Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses. Here, a recombinant replication-deficient adenovirus-based vaccine, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 130 to 230) engineered by codon optimization and tandem repetition for higher-level expression, was constructed and evaluated for its potential as an RSV vaccine in a murine model. A single intranasal immunization with rAd/3xG provided potent protection against RSV challenge which lasted for more than 10 weeks. Strong mucosal immunoglobulin A responses were also induced by a single intranasal immunization but not by intramuscular or oral administration of rAd/3xG. Interestingly, neither gamma interferon- nor interleukin-4-producing CD4 T cells directed to I-E^d-restricted epitope were detected in the lungs of rAd/3xG-immune mice upon challenge, whereas priming with vaccinia virus expressing RSV G (vvG) elicited strong Th1/Th2 mixed CD4 T-cell responses. Lung eosinophilia and vaccine-induced weight loss were significantly lower in the rAd/3xG-immune group than in the vvG-primed group. Together, our data demonstrate that a single intranasal administration of rAd/3xG elicits beneficial protective immunity and represents a promising vaccine regimen against RSV infection.     

Differential role of gamma interferon in inhibiting pulmonary eosinophilia and exacerbating systemic disease in fusion protein-immunized mice undergoing challenge infection with respiratory syncytial virus

Secondary exposure to respiratory syncytial virus (RSV) can lead to immunopathology and enhanced disease in vaccinated individuals. Vaccination with individual RSV proteins influences the type of secondary RSV-specific immune response that develops upon challenge RSV infection, as well as the extent of immunopathology. RSV-specific memory CD4 T cells can directly contribute to immunopathology through their cytokine production. Immunization of BALB/c mice with a recombinant vaccinia virus (vv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia upon RSV challenge, whereas immunization of mice with a vv expressing the fusion (F) protein does not. We analyzed the CD4 T-cell response to an I-E^d-restricted CD4 T-cell epitope within the F protein of RSV corresponding to amino acids 51 to 66 in an effort to better understand the similarities and differences in the immune response elicited by the G versus the F protein. Vaccination with the G protein induces a mixture of RSV G-specific Th1 and Th2 cells with a restricted T-cell receptor repertoire. In contrast, we demonstrate here that immunization with the F protein elicits a broad repertoire of RSV F-specific CD4 T cells that predominantly exhibit a Th1 phenotype. However, in the absence of gamma interferon (IFN-γ), RSV F_51-66-specific CD4 T cells secreted interleukin-5, and mice developed pulmonary eosinophilia after RSV challenge. IFN-γ-deficient mice exhibited decreased weight loss compared to wild-type controls, suggesting that IFN-γ exacerbates systemic disease. These data demonstrate that IFN-γ can have both beneficial and detrimental effects during a secondary RSV infection.     

TLR4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the (299)Gly and (399)Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence ((299)Asp/(399)Thr), or the (299)Gly or (399)Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing (299)Gly or (399)Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-kappaB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-alpha. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus.