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.
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.
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.
The effect of infection history is ignored in most animal models of infectious disease. The attachment protein of respiratory syncytial virus (RSV) induces T helper cell type 2–driven pulmonary eosinophilia in mice similar to that seen in the failed infant vaccinations in the 1960s. We show that previous influenza virus infection of mice: (a) protects against weight loss, illness, and lung eosinophilia; (b) attenuates recruitment of inflammatory cells; and (c) reduces cytokine secretion caused by RSV attachment protein without affecting RSV clearance. This protective effect can be transferred via influenza-immune splenocytes to naive mice and is long lived. Previous immunity to lung infection clearly plays an important and underestimated role in subsequent vaccination and infection. The data have important implications for the timing of vaccinations in certain patient groups, and may contribute to variability in disease susceptibility observed in humans.
viral immunology; murine model; eosinophils; major histocompatibility complex tetramers; mucosal immunology
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.
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.
Cytotoxic T cells; Th1/Th2 T cells; Eosinophils; Lung; Vaccination
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.
CD4-T-helper-cell (Th) differentiation is influenced by costimulatory molecules expressed on conventional dendritic cells (DCs) in regional lymph nodes and results in specific patterns of cytokine production. However, the function of costimulatory molecules on ‘inflammatory’ (CD11b+) DCs in the lung during recall responses is not fully understood, but important for development of novel interventions to limit immunopathological responses to infection. Using a mouse model in which vaccination with vaccinia virus vectors expressing the respiratory syncytial virus (RSV) fusion protein (rVVF) or attachment protein (rVVG) leads to type 1- or type 2-biased cytokine responses respectively upon RSV-challenge, we found expression of CD40 and OX40L on lung inflammatory DCs was higher in rVVF- than in rVVG-primed mice early after RSV-challenge, while the reverse was observed later in the response. Conversely, PD-L2 was higher in rVVG-primed mice throughout. Inflammatory DCs isolated at the resolution of inflammation revealed OX40L on type 1-biased DCs promoted IL-5, while on type 2-biased DCs enhanced IFNγ production by antigen-reactive Th cells. In contrast, PD-L2 promoted IFNγ production irrespective of conditions, suppressing IL-5 only if expressed on type 1-biased DCs. Thus, OX40L and PD-L2 expressed on DCs differentially regulate cytokine production during recall responses in the lung. Manipulation of these costimulatory pathways may provide a novel approach to controlling pulmonary inflammatory responses.
It is known that respiratory syncytial virus (RSV) is the main cause of bronchiolitis and pneumonia in young children. RSV infection often leads to severe acute lung immunopathology, but the underlying immune mechanisms are not yet fully elucidated. Here, we found that RSV infection induced severe acute lung immune injury and promoted the accumulation and activation of lung natural killer (NK) cells at the early stage of infection in BALB/c mice. Activated lung NK cells highly expressed activating receptors NKG2D and CD27 and became functional NK cells by producing a large amount of gamma interferon (IFN-γ), which was responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immune injury and reduced infiltration of total inflammatory cells and production of IFN-γ in bronchoalveolar lavage fluid (BALF). These data show that NK cells are involved in exacerbating the lung immune injury at the early stage of RSV infection via IFN-γ secretion.
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.
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.
Background: Respiratory syncytial virus (RSV) infection can cause bronchial hyperresponsiveness and asthma exacerbations. In mice it results in airway inflammation and airway hyperresponsiveness. Since viral factors influencing these responses are not well defined, a study was undertaken to investigate the role of secreted G protein of human RSV in determining virulence, inflammatory responses, and changes in lung function.
Methods: BALB/c mice were infected with a spontaneous mutant of RSV deficient in secreted G protein (RSV-ΔsG) or with wild type RSV (RSV-WT). Viral titres, numbers of pulmonary inflammatory cells, and concentrations of interferon (IFN)-γ, interleukin (IL)-4, IL-5 and IL-10 in bronchoalveolar lavage (BAL) fluid were determined. Airway function was assessed at baseline and following methacholine provocation using barometric whole body plethysmography.
Result: Following infection with RSV-ΔsG, viral titres were increased 50-fold compared with RSV-WT. Influx of eosinophils and macrophages to the lung and concentrations of IFN-γ and IL-10 in BAL fluid were also significantly higher following infection with RSV-ΔsG. Airway function, both at baseline and after methacholine provocation, was significantly decreased following infection with RSV-ΔsG compared with RSV-WT.
Conclusion: Secreted G protein is likely to be a regulatory factor in RSV infection limiting infectivity of the virus, inflammatory responses in the lungs, and reduction in lung function.
Respiratory syncytial virus (RSV) infection of BALB/c mice previously immunized with a recombinant vaccinia virus (vacv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia, which mimics the response of FI-RSV vaccinated children, as well as increased weight loss, clinical illness, and Penh. We show that RSV infection of eosinophil-deficient mice previously immunized with vacvG results in the development of increased weight loss, clinical illness, and Penh that is similar to wild-type controls. These measures of RSV vaccine-enhanced disease are dependent upon STAT4. Interestingly, neither IL-12 nor IL-23, the two most common STAT4-activating cytokines, proved necessary for the development of disease. We demonstrate that IFN-γ, which is produced following STAT4 activation, contributes to clinical illness and increased Penh, but not weight loss. Our results have important implications for future RSV vaccine design suggesting that enhancing a Th1 response may exacerbate disease.
Rodent; Eosinophils; Lung; Viral; Knockout Mice
Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality. Previous studies have suggested that T-cell responses may contribute to RSV immunopathology, which could be driven by dendritic cells (DCs). DCs are productively infected by RSV, and during RSV infections, there is an increase of DCs in the lungs with a decrease in the blood. Pediatric populations are particularly susceptible to severe RSV infections; however, DC responses to RSV from pediatric populations have not been examined. In this study, primary isolated DCs from cord blood and adult peripheral blood were compared after RSV infection. Transcriptional profiling and biological network analysis identified transforming growth factor beta (TGF-β) and associated signaling molecules as differentially regulated in the two age groups. TGF-β1 was decreased in RSV-infected adult-blood DCs but increased in RSV-infected cord blood DCs. Coculture of adult RSV-infected DCs with autologous T cells induced secretion of gamma interferon (IFN-γ), interleukin 12p70 (IL-12p70), IL-2, and tumor necrosis factor alpha (TNF-α). Conversely, coculture of cord RSV-infected DCs and autologous T cells induced secretion of IL-4, IL-6, IL-1β, and IL-17. Addition of purified TGF-β1 to adult DC-T-cell cocultures reduced secretion of IFN-γ, IL-12p70, IL-2, and TNF-α, while addition of a TGF-β chemical inhibitor to cord DC-T-cell cocultures increased secretion of IL-12p70. These data suggest that TGF-β acts as a major regulator of RSV DC-T-cell responses, which could contribute to immunopathology during infancy.
Respiratory syncytial virus (RSV) is the major cause of infantile bronchiolitis and hospitalization. Severe RSV disease is associated with the development of wheezing in later life. In a mouse model of the delayed effects of RSV, the age at primary infection determines responses to reinfection in adulthood. During primary RSV infection, neonatal BALB/c mice developed only mild disease and recruited CD8 cells that were defective in gamma interferon production. Secondary reinfection of neonatally primed mice caused enhanced inflammation and profuse lung T-cell recruitment. CD4 cell depletion during secondary RSV challenge attenuated disease (measured by weight loss); depletion of CD8 cells also markedly attenuated disease severity but enhanced lung eosinophilia, and depletion of both CD4 and CD8 cells together completely abrogated weight loss. Depletion of CD8 (but not CD4) cells during primary neonatal infection was protective against weight loss during adult challenge. Therefore, T cells, in particular CD8 T cells, play a central role in the outcome of neonatal infection by enhancing disease during secondary challenge. These findings demonstrate a crucial role for T cells in the regulation of immune responses after neonatal infection.
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.
Respiratory syncytial virus (RSV) is the main cause of bronchiolitis, the major cause of hospitalization of infants. An ideal RSV vaccine would be effective for neonates, but the immune responses of infants differ markedly from those of adults, often showing a bias toward T-helper 2 (Th2) responses and reduced gamma interferon (IFN-γ) production. We previously developed recombinant RSV vectors expressing IFN-γ and interleukin-4 (IL-4) that allow us to explore the role of these key Th1 and Th2 cytokines during infection. The aim of the current study was to explore whether an immunomodulation of infant responses could enhance protection. The expression of IFN-γ by a recombinant RSV vector (RSV/IFN-γ) attenuated primary viral replication in newborn mice without affecting the development of specific antibody or T-cell responses. Upon challenge, RSV/IFN-γ mice were protected from the exacerbated disease observed for mice primed with wild-type RSV; however, antiviral immunity was not enhanced. Conversely, the expression of IL-4 by recombinant RSV did not affect virus replication in neonates but greatly enhanced Th2 immune responses upon challenge without affecting weight loss. These studies demonstrate that it is possible to manipulate infant immune responses by using cytokine-expressing recombinant viruses and that neonatal deficiency in IFN-γ responses may lead to enhanced disease during secondary infection.
Respiratory syncytial virus (RSV) is a major viral pathogen of infants and the elderly. Significant morbidity is caused by an overexuberant mixed lung cell infiltrate, which is thought to be driven by chemokines. One of the main chemotactic mediators responsible for the movement of eosinophils is CCL11 (eotaxin). Using a mouse model of eosinophilic bronchiolitis induced by RSV, we show here that treatment in vivo with a blocking antibody to CCL11 greatly reduces lung eosinophilia and disease severity. In addition, anti-CCL11 caused a striking inhibition of CD4-T-cell influx and shifted cytokine production away from interleukin-5 without reducing the resistance to viral replication. These results suggest that in addition to influencing eosinophil diapedesis and survival, anti-CCL11 has an action on T cells. These studies strengthen the case for anti-CCL11 treatment of Th2-driven diseases.
In BALB/c mice, sensitization to respiratory syncytial virus (RSV) attachment (G) glycoprotein leads to the development of lung eosinophilia upon challenge infection with RSV, a pathology indicative of a strong in vivo induction of a Th-2-type response. In this study, we found that a strong, RSV G-specific, Th-1-type cytokine response occurred simultaneously with a Th-2-type response in G-primed mice after RSV challenge. Both Th-1 and Th-2 effector CD4+ T cells recognized a single immunodominant site on this protein, implying that the differentiation of memory CD4+ T cells along the Th-1 or Th-2 effector pathway was independent of the epitope specificity of the T cells. A similar observation was made in G-primed H-2b haplotype mice after RSV challenge, further suggesting that this process is not dependent on the peptide epitope presented. On the other hand, genes mapping to loci outside of the major histocompatibility complex region are crucial regulators of the development of a Th-2-type response and lung eosinophilia. The implication of these findings for the immune mechanisms underlying the pathogenesis of RSV is discussed.
We analyzed the immune responses evoked by a series of overlapping peptides to better understand the molecular basis for respiratory syncytial virus (RSV) G protein–induced eosinophilia in BALB/c mice. In vitro stimulation of spleen cells from natural G protein–primed mice showed dominant proliferative and cytokine (interferon [IFN]-γ and interleukin [IL]-5) responses to a peptide encompassing amino acids 184–198. Mice vaccinated with peptide 184– 198 conjugated to keyhole limpet hemocyanin showed significant pulmonary eosinophilia (39.5%) after challenge with live RSV. In contrast, mice immunized with a peptide (208–222) conjugate associated with induction of IFN-γ secreting spleen cells did not exhibit pulmonary eosinophilia after challenge. The in vivo depletion of CD4+ cells abrogated pulmonary eosinophilia in mice vaccinated with the peptide 184–198 conjugate, whereas the depletion of CD8+ cells had a negligible effect. Therefore, we have identified an association between peptide 184– 198 of natural G protein and the CD4+ T cell–mediated induction of pulmonary eosinophilia after live RSV challenge. Out of 43 human donors, 6 provided peripheral blood mononuclear cells that showed reactivity to G protein from RSV A2, 3 of which responded to peptide 184– 198. The results have important implications for the development of a vaccine against RSV.
respiratory syncytial virus; G protein; eosinophilia; T helper cell; peptide
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-α/β) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-α/β receptor.
Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon γ after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit β-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.
respiratory syncytial virus; DNA vaccine; vector design; F protein; immune modulation
Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2Kd-restricted CD8+ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.
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.
BALB/c mice sensitized to vaccinia virus expressed G protein of respiratory syncytial virus (RSV) develop a Th2-type cytokine response and pulmonary eosinophilia when challenged with live RSV. In this study, BALB/c mice were immunized or challenged with an RSV mutant lacking the G and SH proteins or with DNA vaccines coding for RSV G or F protein. F or G protein DNA vaccines were capable of sensitizing for pulmonary eosinophilia. The absence of the G and/or SH protein in the infecting virus resulted in a consistent increase both in pulmonary natural killer cells and in gamma interferon and tumor necrosis factor expression, as well as, with primary infection, a variable increase in neutrophils and CD11b+ cells. The development of pulmonary eosinophilia in formalin-inactivated RSV-vaccinated mice required the presence of the G and/or SH protein in the challenge virus. These data show that G and/or SH protein has a marked impact on the inflammatory and innate immune response to RSV infection.