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1.  Vβ14+ T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV 
Journal of Virology  2004;78(16):8753-8760.
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 Vβ14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vβ14+ 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 Vβ14 component of the TCR. vvGs-immunized mice treated with anti-Vβ14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vβ14+ T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vβ chain usage confirmed a bias toward Vβ14 expression on CD4+ T cells from vvGs-immunized mice, whereas the CD4+ T cells in FI-RSV-immunized mice expressed a diverse array of Vβ 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.
doi:10.1128/JVI.78.16.8753-8760.2004
PMCID: PMC479099  PMID: 15280483
2.  Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection  
T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8+ T cytolytic T cells on the differentiation and activation of functionally distinct CD4+ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8+ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8+ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8+ T cells in this process was further supported by the results in CD8+ T cell deficient, β2 microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8+ T cell compartment. These results indicate that CD8+ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.
PMCID: PMC2198992  PMID: 9236194
3.  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▿  
Journal of Virology  2007;82(5):2196-2207.
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-Ed-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 F51-66-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.
doi:10.1128/JVI.01949-07
PMCID: PMC2258940  PMID: 18094193
4.  Pulmonary Vγ4+ γδ T Cells Have Proinflammatory and Antiviral Effects in Viral Lung Disease1 
Host defenses, while effecting viral clearance, contribute substantially to inflammation and disease. This double action is a substantial obstacle to the development of safe and effective vaccines against many agents, particularly respiratory syncytial virus (RSV). RSV is a common cold virus and the major cause of infantile bronchiolitis worldwide. The role of αβ T cells in RSV-driven immunopathology is well studied, but little is known about the role of “unconventional” T cells. During primary RSV challenge of BALB/c mice, some Vγ7+ γδ T cells were present; however, immunization with a live vaccinia vector expressing RSV F protein substantially enhanced Vγ4+ γδ T cell influx after RSV infection. Harvested early, these cells produced IFN-γ, TNF, and RANTES after ex vivo stimulation. By contrast, those recruited 5 days after challenge made IL-4, IL-5, and IL-10. Depletion of γδ T cells in vivo reduced lung inflammation and disease severity and slightly increased peak viral replication but did not prevent viral clearance. These studies demonstrate a novel role for γδ T cells in the development of immunopathology and cellular influx into the lungs after immunization and RSV challenge. Though a minor population, γδ T cells have a critical influence on disease and are an attractive interventional target in the alleviation of viral lung disease.
PMCID: PMC2646375  PMID: 19124761
5.  Respiratory Syncytial Virus (RSV) G Glycoprotein Is Not Necessary for Vaccine-Enhanced Disease Induced by Immunization with Formalin-Inactivated RSV 
Journal of Virology  2004;78(11):6024-6032.
Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.
doi:10.1128/JVI.78.11.6024-6032.2004
PMCID: PMC415805  PMID: 15141000
6.  Development of Safe and Effective RSV Vaccine by Modified CD4 Epitope in G Protein Core Fragment (Gcf) 
PLoS ONE  2014;9(4):e94269.
Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183–195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51–66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.
doi:10.1371/journal.pone.0094269
PMCID: PMC3988050  PMID: 24736750
7.  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. 
Journal of Virology  1996;70(5):2852-2860.
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.
PMCID: PMC190142  PMID: 8627759
8.  IL-17A inhibits airway reactivity induced by RSV infection during allergic airway inflammation 
Thorax  2013;68(8):717-723.
Background
Viral infections are the most frequent cause of asthma exacerbations and are linked to increased airway reactivity (AR) and inflammation. Mice infected with respiratory syncytial virus (RSV) during ovalbumin (OVA)-induced allergic airway inflammation (OVA/RSV) had increased AR compared to OVA or RSV mice alone. Further, IL-17A was only increased in OVA/RSV mice.
Objective
To determine if IL-17A increases AR and inflammation in the OVA/RSV model.
Methods
Wild-type BALB/c and IL-17A KO mice underwent mock, RSV, OVA, or OVA/RSV protocols. Lungs, bronchoalveolar lavage (BAL) fluid, and/or mediastinal lymph nodes (MLNs) were harvested post infection. Cytokine expression was determined by flow cytometry and ELISA in the lungs or BAL fluid. MLNs were restimulated with either OVA (323–229) peptide or RSV M2 (127–135) peptide and IL-17A protein expression was analyzed. AR was determined by methacholine challenge.
Results
RSV increased IL-17A protein expression by OVA-specific T cells 6 days post infection. OVA/RSV mice had decreased IFN-α and IFN-β protein expression compared to RSV mice. OVA/RSV mice had increased IL-23 mRNA expression in lung homogenates compared to mock, OVA, or RSV mice. Unexpectedly, IL-17A KO OVA/RSV mice had increased AR compared to WT OVA/RSV mice. Further, IL-17A KO OVA/RSV mice had increased eosinophils, lymphocytes, and IL-13 protein expression in BAL fluid compared to WT OVA/RSV mice.
Conclusions
IL-17A negatively regulated AR and airway inflammation in OVA/RSV mice. This finding is important because IL-17A has been identified as a potential therapeutic target in asthma, and inhibiting IL-17A in the setting of virally induced asthma exacerbations may have adverse consequences.
doi:10.1136/thoraxjnl-2012-202404
PMCID: PMC3916091  PMID: 23422214
IL-17A; airway reactivity; CD4+ T cells; allergic inflammation; RSV
9.  Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice 
Journal of Virology  2013;87(20):10946-10954.
During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3DTR mice (which allow specific conditional depletion of Foxp3+ T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4+ and CD8+ T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4+ T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.
doi:10.1128/JVI.01295-13
PMCID: PMC3807299  PMID: 23926350
10.  Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection 
PLoS Medicine  2013;10(11):e1001549.
In this study, Mejias and colleagues found that specific blood RNA profiles of infants with RSV LRTI allowed for specific diagnosis, better understanding of disease pathogenesis, and better assessment of disease severity.
Please see later in the article for the Editors' Summary
Background
Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity.
Methods and Findings
This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (<6 mo). We identified a genomic score that significantly correlated with outcomes of care including a clinical disease severity score and, more importantly, length of hospitalization and duration of supplemental O2.
Conclusions
Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lower respiratory tract infections (LRTIs)—bacterial and viral infections of the lungs and airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for 14% of all child deaths. The leading cause of viral LTRIs in children is respiratory syncytial virus (RSV), which is readily transmitted from person to person by direct contact with nasal fluids or airborne droplets. Almost all children have an RSV infection before their second birthday, but most have only minor symptoms similar to those of a common cold and are cared for at home. Unfortunately, some children develop more serious conditions when they become infected with RSV, such as pneumonia or bronchiolitis (swelling and mucus build-up in the bronchioles, the smallest air passages in the lungs). These children have to be admitted to the hospital for supportive care—there is no specific treatment for RSV infection—such as the provision of supplemental oxygen.
Why Was This Study Done?
The lack of a treatment (and of a vaccine) for RSV is largely due to our incomplete understanding of the cellular events and reactions, including the host immune response, that occur during the development of an RSV infection (disease pathogenesis). Moreover, based on physical examination and available diagnostic tools, it is impossible to predict which children infected with RSV will develop a serious condition that requires hospitalization and which ones can be safely nursed at home. Here, the researchers use microarrays to analyze the global host response to acute RSV LTRI in infants, to define gene expression patterns that are specific to RSV infection rather than infection with other common respiratory viruses, and to identify biomarkers that indicate the severity of RSV infection. “Microarray” analysis allows researchers to examine gene expression patterns (“RNA transcriptional profiles”) in, for example, whole blood; a biomarker is a molecule whose level in bodily fluids or tissues indicates how a disease might develop and helps with patient classification.
What Did the Researchers Do and Find?
The researchers compared the RNA transcriptional profile in whole blood taken from children less than two years old hospitalized with RSV, human rhinovirus, or influenza virus infection (rhinovirus and influenza are two additional viral causes of LRTI), and from healthy infants. Using “statistical group comparisons,” they identified more than 2,000 transcripts that were differentially expressed in blood from 45 infants with RSV infection and from 14 healthy matched controls. Genes related to interferon function (interferons are released by host cells in response to the presence of disease-causing organisms) and neutrophil function (neutrophils are immune system cells that, like interferons, are involved in the innate immune response, the body's first line of defense against infection) were among the most overexpressed genes in infants infected with RSV. Genes regulating T and B cells (components of the adaptive immune response, the body's second-line of defense against infection) were among the most underexpressed genes. This specific transcriptional profile, which was validated in three additional groups of infants, accurately distinguished between infants infected with RSV and those infected with human rhinovirus or influenza virus. Finally, a “molecular distance to health” score (a numerical score that quantifies the transcriptional perturbation associated with an illness) was correlated with the clinical disease severity score of the study participants, with how long they needed supplemental oxygen, and with their duration of hospitalization.
What Do These Findings Mean?
These findings suggest that it might be possible to use whole blood RNA transcriptional profiles to distinguish between infants infected with RSV and those with other viruses that commonly cause LRTI. Moreover, if these findings can be replicated in more patients (including non-hospitalized children), gene expression profiling might provide a strategy for triaging patients with RSV infections when they first present to an emergency department and for monitoring clinical changes during the course of the infection, particularly given the development of molecular tools that might soon enable the “real time” acquisition of transcriptional profiles in the clinical setting. Finally, although certain aspects of the study design limit the accuracy and generalizability of the study's findings, these data provide new insights into the pathogenesis of RSV infection and open new avenues for the discovery of biomarkers for RSV infection and for the identification of therapeutic and preventative targets.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001549.
This study is further discussed in a PLOS Medicine Perspective by Peter Openshaw
The US Centers for Disease Control and Prevention provides information about RSV infection
The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about RSV infections
The UK National Health Service Choices website provides information about bronchiolitis
The British Lung Foundation also provides information on RSV and on bronchiolitis
MedlinePlus provides links to other resources about RSV infections and about pneumonia (in English and Spanish); the MedlinePlus encyclopedia has a page on bronchiolitis (in English and Spanish)
PATH is an international non-profit organization investigating new RSV vaccines
doi:10.1371/journal.pmed.1001549
PMCID: PMC3825655  PMID: 24265599
11.  An adjuvanted respiratory syncytial virus fusion protein induces protection in aged BALB/c mice 
Background
Respiratory Syncytial Virus (RSV) causes significant disease in the elderly, in part, because immunosenescence impairs protective immune responses to infection in this population. Despite previous and current efforts, there is no RSV vaccine currently licensed in infants or elderly adults. Adjuvanted RSV subunit vaccines have the potential to boost waning immune responses and reduce the burden of RSV disease in the elderly population.
Results
We used an aged BALB/c mouse model to evaluate immune responses to RSV Fusion (F) protein in the absence and presence of an alum adjuvant. We demonstrate that aged BALB/c mice immunized with alum-adjuvanted RSV F protein had significantly reduced lung viral titers at day 4 following challenge with wild-type (wt) RSV. Serum neutralizing antibody titers measured on day 27 correlated with protection in both young and aged vaccinated mice, although the magnitude of antibody titers was lower in aged mice. Unlike young mice, in aged mice, alum-adjuvanted RSV F did not induce lung TH2-type cytokines or eosinophil infiltration compared to non-adjuvanted F protein following wt RSV challenge.
Conclusion
Our studies demonstrate that neutralizing anti-RSV antibody titers correlate with protection in both young and aged BALB/c mice vaccinated with RSV F protein vaccines. The F + alum formulation mediated greater protection compared to the non-adjuvanted F protein in both young and aged mice. However, while alum can boost F-specific antibody responses in aged mice, it does not completely overcome the reduced ability of a senescent immune system to respond to the RSV F antigen. Thus, our data suggest that a stronger adjuvant may be required for the prevention of RSV disease in immunosenescent populations, to achieve the appropriate balance of protective neutralizing antibodies and effective TH1-type cytokine response along with minimal lung immunopathology.
doi:10.1186/1742-4933-9-21
PMCID: PMC3534329  PMID: 23031690
Respiratory Syncytial Virus; Immunosenescence; Alum; Adjuvant; Aged mice
12.  Endogenous IL-21 regulates pathogenic mucosal CD4 T-cell responses during enhanced RSV disease in mice 
Mucosal Immunology  2012;6(4):704-717.
A role for interleukin-21 (IL-21) has recently been found in several diseases, but contribution to mucosal defences has not been described. In BALB/c mice infected with respiratory syncytial virus (RSV), IL-21 depletion had little effect in primary infection. However, depletion of mice during priming with recombinant vaccinia expressing RSV G protein (which primes RSV-specific T helper type 2 cells and causes lung eosinophilia during RSV infection) further exacerbated pathology during RSV challenge, with reduced viral clearance and impaired virus-specific serum antibody responses. This enhancement was accompanied by lymphocyte, neutrophil, and antigen-presenting cell recruitment to the lungs, with increased bronchoalveolar lavage interferon-γ and IL-17 levels. Adoptive transfer of splenic CD4 T cells from depleted mice into naive recipients replicated these effects, indicating that IL-21 mediates its effects via CD4 T cells. Endogenous IL-21, therefore, has potent and specific effects on mucosal antiviral responses, assisting viral clearance, regulating pulmonary T- and B-cell responses, and inhibiting IL-17 production.
doi:10.1038/mi.2012.108
PMCID: PMC3610641  PMID: 23168836
13.  Differential Regulation of GM1 and Asialo-GM1 Expression by T Cells and Natural Killer (NK) Cells in Respiratory Syncytial Virus Infection 
Viral Immunology  2008;21(3):327-339.
Abstract
We previously reported that respiratory syncytial virus (RSV) infection increases lung CD8+ T cell GM1 expression. The related lipid asialo-GM1 (ASGM1) is expressed by T cells in viral infection and by natural killer (NK) cells. The in vivo co-expression of GM1 and ASGM1 by immune cells is not defined. Here we analyzed lung lymphocyte GM1 and ASGM1 expression in RSV-infected mice. GM1 and ASGM1 were coordinately up-regulated by activated CD8+ T cells in RSV-infected BALB/c and C57BL/6 mice. In contrast, RSV infection had no effect on constitutively high NK cell GM1 expression, while increasing NK cell ASGM1 expression. GM1 and ASGM1 co-localized in lipid raft structures in NK and CD8+ T cells sorted from the lungs of RSV-infected mice. Anti-ASGM1 Ab treatment of RSV-infected BALB/c mice depleted GM1/ASGM1-expressing NK cells and GM1/ASGM1-expressing T cells, reduced lung IFN-γ levels, increased viral load, delayed viral clearance, and reduced illness. STAT1–/– mice are more susceptible to RSV replication and disease than wild-type mice. In RSV-infected STAT1–/– mice, anti-ASGM1 Ab altered cytokine levels, but in contrast to BALB/c mice, antibody treatment had no effect on viral load or illness. Taken together, GM1 and ASGM1 expression are differentially regulated by T and NK cells in RSV infection. Also, GM1/ASGM1-expressing cells are important for control of RSV in BALB/c mice, whereas STAT1–/– mice clear RSV by an alternative pathway.
doi:10.1089/vim.2008.0003
PMCID: PMC2952133  PMID: 18788941
14.  Secreted Respiratory Syncytial Virus G Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism 
Journal of Virology  1999;73(10):8485-8495.
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.
PMCID: PMC112868  PMID: 10482601
15.  STAT1 negatively regulates lung basophil IL-4 expression induced by respiratory syncytial virus infection1 
IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3−CD49b+ cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3−CD49b+FcεRI+c-kit−). Because STAT1−/− mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1−/− mice. RSV infection resulted in significantly more IL-4-expressing CD3−CD49b+ cells in the lungs of STAT1−/− mice than in BALB/c mice. CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1−/− mice. Depletion of basophils in RSV-infected STAT1−/− mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.
doi:10.4049/jimmunol.0803167
PMCID: PMC3755459  PMID: 19587017
basophils; cytokines; viral; lung
16.  The Number of RSV-Specific Memory CD8 T Cells in the Lung is Critical for their Ability to Inhibit Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Pulmonary Eosinophilia1 
Children that were administered a formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine experienced enhanced respiratory disease, including pulmonary eosinophilia, after contracting a natural RSV infection. RSV vaccine-enhanced disease can be mimicked in BALB/c mice immunized with either FI-RSV or with a recombinant vaccinia virus (vacv) expressing the RSV attachment (G) protein. We have recently demonstrated that memory CD8 T cells directed against the RSV immunodominant M282−90 epitope inhibit the development of pulmonary eosinophilia in either vacvG- or FI-RSV-immunized mice by reducing the total number of Th2 cells in the lung after RSV challenge. Here we show that memory CD8 T cells specific to a subdominant epitope within the RSV fusion (F) protein fail to inhibit the development of pulmonary eosinophilia after RSV challenge of mice previously co-immunized with vacvF and with either vacvG or FI-RSV. We observed that the inability of RSV F85-specific memory CD8 T cells to inhibit the development of pulmonary eosinophilia was largely due to an inadequate total number of F85-specific memory CD8 T cells in the lung at early times after RSV challenge. Increasing the number of F85-specific memory CD8 T cells after immunization grants them the ability to inhibit RSV vaccine-enhanced pulmonary eosinophilia. Moreover, we demonstrate that RSV-specific memory CD8 T cells, when present in sufficient numbers, inhibit the production of the Th2-associated chemokines CCL17 and CCL22. Taken together, these results indicate that RSV-specific memory CD8 T cells may alter the trafficking of Th2 cells and eosinophils into the lung.
PMCID: PMC2587004  PMID: 19017987
Cytotoxic T cells; Th1/Th2 T cells; Eosinophils; Lung; Vaccination
17.  Association between Respiratory Syncytial Virus Activity and Pneumococcal Disease in Infants: A Time Series Analysis of US Hospitalization Data 
PLoS Medicine  2015;12(1):e1001776.
Daniel Weinberger and colleagues examine a possible interaction between two serious respiratory infections in children under 2 years of age.
Please see later in the article for the Editors' Summary
Background
The importance of bacterial infections following respiratory syncytial virus (RSV) remains unclear. We evaluated whether variations in RSV epidemic timing and magnitude are associated with variations in pneumococcal disease epidemics and whether changes in pneumococcal disease following the introduction of seven-valent pneumococcal conjugate vaccine (PCV7) were associated with changes in the rate of hospitalizations coded as RSV.
Methods and Findings
We used data from the State Inpatient Databases (Agency for Healthcare Research and Quality), including >700,000 RSV hospitalizations and >16,000 pneumococcal pneumonia hospitalizations in 36 states (1992/1993–2008/2009). Harmonic regression was used to estimate the timing of the average seasonal peak of RSV, pneumococcal pneumonia, and pneumococcal septicemia. We then estimated the association between the incidence of pneumococcal disease in children and the activity of RSV and influenza (where there is a well-established association) using Poisson regression models that controlled for shared seasonal variations. Finally, we estimated changes in the rate of hospitalizations coded as RSV following the introduction of PCV7. RSV and pneumococcal pneumonia shared a distinctive spatiotemporal pattern (correlation of peak timing: ρ = 0.70, 95% CI: 0.45, 0.84). RSV was associated with a significant increase in the incidence of pneumococcal pneumonia in children aged <1 y (attributable percent [AP]: 20.3%, 95% CI: 17.4%, 25.1%) and among children aged 1–2 y (AP: 10.1%, 95% CI: 7.6%, 13.9%). Influenza was also associated with an increase in pneumococcal pneumonia among children aged 1–2 y (AP: 3.2%, 95% CI: 1.7%, 4.7%). Finally, we observed a significant decline in RSV-coded hospitalizations in children aged <1 y following PCV7 introduction (−18.0%, 95% CI: −22.6%, −13.1%, for 2004/2005–2008/2009 versus 1997/1998–1999/2000). This study used aggregated hospitalization data, and studies with individual-level, laboratory-confirmed data could help to confirm these findings.
Conclusions
These analyses provide evidence for an interaction between RSV and pneumococcal pneumonia. Future work should evaluate whether treatment for secondary bacterial infections could be considered for pneumonia cases even if a child tests positive for RSV.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Respiratory infections—bacterial and viral infections of the lungs and the airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for about 15% of all child deaths. The leading cause of bacterial pneumonia in children is Streptococcus pneumoniae, which is transmitted through contact with infected respiratory secretions. S. pneumoniae usually causes noninvasive diseases such as bronchitis, but sometimes the bacteria invade the lungs, the bloodstream, or the covering of the brain, where they cause pneumonia, septicemia, or meningitis, respectively. These potentially fatal invasive pneumococcal diseases can be treated with antibiotics but can also be prevented by vaccination with pneumococcal conjugate vaccines such as PCV7. The leading cause of viral pneumonia is respiratory syncytial virus (RSV), which is also readily transmitted through contact with infected respiratory secretions. Almost all children have an RSV infection before their second birthday—RSV usually causes a mild cold-like illness. However, some children infected with RSV develop pneumonia and have to be admitted to hospital for supportive care such as the provision of supplemental oxygen; there is no specific treatment for RSV infection.
Why Was This Study Done?
Co-infections with bacteria and viruses can sometimes have a synergistic effect and lead to more severe disease than an infection with either type of pathogen (disease-causing organism) alone. For example, influenza infections increase the risk of invasive pneumococcal disease. But does pneumococcal disease also interact with RSV infection? It is important to understand the interaction between pneumococcal disease and RSV to improve the treatment of respiratory infections in young children, but the importance of bacterial infections following RSV infection is currently unclear. Here, the researchers undertake a time series analysis of US hospitalization data to investigate the association between RSV activity and pneumococcal disease in infants. Time series analysis uses statistical methods to analyze data collected at successive, evenly spaced time points.
What Did the Researchers Do and Find?
For their analysis, the researchers used data collected between 1992/1993 and 2008/2009 by the State Inpatient Databases on more than 700,000 hospitalizations for RSV and more than 16,000 hospitalizations for pneumococcal pneumonia or septicemia among children under two years old in 36 US states. Using a statistical technique called harmonic regression to measure seasonal variations in disease incidence (the rate of occurrence of new cases of a disease), the researchers show that RSV and pneumococcal pneumonia shared a distinctive spatiotemporal pattern over the study period. Next, using Poisson regression models (another type of statistical analysis), they show that RSV was associated with significant increases (increases unlikely to have happened by chance) in the incidence of pneumococcal disease. Among children under one year old, 20.3% of pneumococcal pneumonia cases were associated with RSV activity; among children 1–2 years old, 10.1% of pneumococcal pneumonia cases were associated with RSV activity. Finally, the researchers report that following the introduction of routine vaccination in the US against S. pneumoniae with PCV7 in 2000, there was a significant decline in hospitalizations for RSV among children under one year old.
What Do These Findings Mean?
These findings provide evidence for an interaction between RSV and pneumococcal pneumonia and indicate that RSV is associated with increases in the incidence of pneumococcal pneumonia, particularly in young infants. Notably, the finding that RSV hospitalizations declined after the introduction of routine pneumococcal vaccination suggests that some RSV hospitalizations may have a joint viral–bacterial etiology (cause), although it is possible that PCV7 vaccination reduced the diagnosis of RSV because fewer children were hospitalized with pneumococcal disease and subsequently tested for RSV. Because this is an ecological study (an observational investigation that looks at risk factors and outcomes in temporally and geographically defined populations), these findings do not provide evidence for a causal link between hospitalizations for RSV and pneumococcal pneumonia. The similar spatiotemporal patterns for the two infections might reflect another unknown factor shared by the children who were hospitalized for RSV or pneumococcal pneumonia. Moreover, because pooled hospitalization discharge data were used in this study, these results need to be confirmed through analysis of individual-level, laboratory-confirmed data. Importantly, however, these findings support the initiation of studies to determine whether treatment for bacterial infections should be considered for children with pneumonia even if they have tested positive for RSV.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001776.
The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about pneumonia
The US Centers for Disease Control and Prevention provides information on all aspects of pneumococcal disease and pneumococcal vaccination, including personal stories and information about RSV infection
The UK National Health Service Choices website provides information about pneumonia (including a personal story) and about pneumococcal diseases
KidsHealth, a website provided by the US-based non-profit Nemours Foundation, includes information on pneumonia and on RSV (in English and Spanish)
MedlinePlus provides links to other resources about pneumonia, RSV infections, and pneumococcal infections (in English and Spanish)
HCUPnet provides aggregated hospitalization data from the State Inpatient Databases used in this study
doi:10.1371/journal.pmed.1001776
PMCID: PMC4285401  PMID: 25562317
18.  Resveratrol Inhibits the TRIF-Dependent Pathway by Upregulating Sterile Alpha and Armadillo Motif Protein, Contributing to Anti-Inflammatory Effects after Respiratory Syncytial Virus Infection 
Journal of Virology  2014;88(8):4229-4236.
ABSTRACT
Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in young children and the leading cause of infant hospitalization worldwide. Uncontrolled response to RSV is mediated by a toll-like receptor (TLR)-mediated immune response. Resveratrol possesses anti-RSV activity and is an inhibitor of the TRIF/TBK1/IRF-3 complex. We hypothesize that resveratrol inhibits the TRIF-dependent pathway through upregulation of SARM post-RSV infection. BALB/c mice were infected with RSV and were injected with resveratrol 1 h postinoculation. SARM short interfering RNA was administered to RSV-infected and resveratrol-treated mice. Lung function was measured by whole-body plethysmography, lung histopathology was examined, and lymphocytes in bronchoalveolar lavage fluid were quantified. SARM and TRIF protein expression were detected in the lung by Western blot analyses. The expression of gamma interferon in bronchoalveolar lavage fluid (BALF) was evaluated by enzyme-linked immunosorbent assay (ELISA). SARM expression was reduced and TRIF expression was increased after infection with RSV. Resveratrol increased SARM expression and decreased TRIF expression after RSV infection. SARM knockdown in resveratrol-treated mice enhanced gamma interferon production, RSV-induced airway inflammation, and airway hyperresponsiveness (AHR). Resveratrol decreased TRIF expression and prevented the RSV-mediated reduction of SARM expression. Resveratrol-mediated inhibition of the TRIF-dependent pathway may be dependent on SARM expression.
IMPORTANCE Our study provides insights into the regulation of innate immunity in response to RSV infection. The results suggest that resveratrol-mediated alterations in SARM have therapeutic potential against RSV immunopathology caused by deregulation of the TLR-mediated immune response. Ultimately, improved insight into the complex interplay between TLR adaptor proteins and the occurrence of severe RSV infection might lead to novel therapeutic treatment strategies, such as TLR adjuvants.
doi:10.1128/JVI.03637-13
PMCID: PMC3993725  PMID: 24478430
19.  Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge 
Journal of Virology  1998;72(4):2871-2880.
The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4+ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.
PMCID: PMC109732  PMID: 9525607
20.  Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease 
Immune Network  2012;12(1):8-17.
Background
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile.
Methods
We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G.
Results
Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection.
Conclusion
Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.
doi:10.4110/in.2012.12.1.8
PMCID: PMC3329602  PMID: 22536165
Respiratory syncytial virus; Glycoprotein; Recombinant baculovirus; Protective immunity
21.  Autocrine Regulation of Pulmonary Inflammation by Effector T-Cell Derived IL-10 during Infection with Respiratory Syncytial Virus 
PLoS Pathogens  2011;7(8):e1002173.
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.
Author Summary
IL-10 is a major anti-inflammatory protein that plays an essential role in regulating the balance between pathogen clearance by the immune response and immune mediated injury resulting from the immune response to pathogen infection. In this report, we demonstrate that anti-viral effector T cells, a critical cell type responsible for respiratory syncytial virus clearance, are able to produce a large quantity of IL-10. The function of IL-10 is to control the immune response in order to avoid the development of excessive pulmonary inflammation associated with the clearance of infectious virus. We further identified a likely mechanism that T cell-derived IL-10 operates to control inflammation and describe a novel potential target of IL-10 action in the RSV infected lungs. Our data thus may lay the ground for the future studies exploring the application of IL-10 in therapeutic approaches to modulate pulmonary inflammation and injury in young infants suffering severe respiratory syncytial virus induced diseases.
doi:10.1371/journal.ppat.1002173
PMCID: PMC3150291  PMID: 21829368
22.  Respiratory Syncytial Virus Reverses Airway Hyperresponsiveness to Methacholine in Ovalbumin-Sensitized Mice 
PLoS ONE  2012;7(10):e46660.
Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2–8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M3-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations.
doi:10.1371/journal.pone.0046660
PMCID: PMC3462783  PMID: 23056391
23.  Pulmonary Eosinophilia Is Attenuated by Early Responding CD8+ Memory T Cells in a Murine Model of RSV Vaccine-Enhanced Disease 
Viral immunology  2009;22(4):243-251.
Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4+ T-cell response to challenge infection with some features characteristic of Th-2 CD4+ T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8+ T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8+ memory T-cell (CD8+ Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8+ T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8+ Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8+ T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8+ Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8+ T cells regulate this process is not by modulating the differentiation or development of the CD4+ Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8+ Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.
doi:10.1089/vim.2009.0016
PMCID: PMC2885249  PMID: 19594395
24.  Pulmonary Eosinophilia Is Attenuated by Early Responding CD8+ Memory T Cells in a Murine Model of RSV Vaccine-Enhanced Disease 
Viral Immunology  2009;22(4):243-251.
Abstract
Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4+ T-cell response to challenge infection with some features characteristic of Th-2 CD4+ T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8+ T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8+ memory T-cell (CD8+ Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8+ T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8+ Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8+ T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8+ Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8+ T cells regulate this process is not by modulating the differentiation or development of the CD4+ Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8+ Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.
doi:10.1089/vim.2009.0016
PMCID: PMC2885249  PMID: 19594395
25.  Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine 
Journal of Virology  2014;88(15):8597-8614.
ABSTRACT
Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants.
IMPORTANCE Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.
doi:10.1128/JVI.00983-14
PMCID: PMC4135953  PMID: 24850731

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