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

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

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Combination Therapy Using Monoclonal Antibodies against Respiratory Syncytial Virus (RSV) G Glycoprotein Protects from RSV Disease in BALB/c Mice

Therapeutic options to control respiratory syncytial virus (RSV) are limited, thus development of new therapeutics is high priority. Previous studies with a monoclonal antibody (mAb) reactive to an epitope proximal to the central conserved region (CCR) of RSV G protein (mAb 131-2G) showed therapeutic efficacy for reducing pulmonary inflammation RSV infection in BALB/c mice. Here, we show a protective effect in RSV-infected mice therapeutically treated with a mAb (130-6D) reactive to an epitope within the CCR of G protein, while treatment with a mAb specific for a carboxyl G protein epitope had no effect. Combined treatment with mAbs 130-6D and 131-2G significantly decreased RSV-associated pulmonary inflammation compared to either antibody alone. The results suggest that anti-RSV G protein mAbs that react at or near the CCR and can block RSV G protein-mediated activities are effective at preventing RSV disease and may be an effective strategy for RSV therapeutic treatment.     

Decrease in Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice

Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study.     

Recombinant Respiratory Syncytial Virus (RSV) Bearing a Set of Mutations from Cold-Passaged RSV Is Attenuated in Chimpanzees

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (cp) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.     

Respiratory Syncytial Virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice

Background

Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity.

Methods

Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood.

Results

RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes.

Conclusions

RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.     

Cytotoxic T-Lymphocyte Precursor Frequencies in BALB/c Mice after Acute Respiratory Syncytial Virus (RSV) Infection or Immunization with a Formalin-Inactivated RSV Vaccine

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.Respiratory syncytial virus (RSV) infection in infants and young children often results in lower respiratory tract disease and is a high priority for vaccine development (1, 2). Attempts to develop an effective live, inactivated, or subunit vaccine have been unsuccessful (24, 25, 28). Early efforts at vaccinating young children with a formalin-inactivated RSV (FI-RSV) vaccine failed to protect the children from naturally acquired infection and actually enhanced lower respiratory tract disease upon later virus infection (2, 15, 24, 25). This enhanced disease has created concern about the safety of any nonlive RSV vaccine and, consequently, understanding the pathogenesis of FI-RSV-induced enhanced disease is critically important to vaccine development. Studies with BALB/c mice suggest that induction of memory T cells producing Th2-like cytokines, as a result of FI-RSV vaccination, may be key to the pathogenesis of enhanced disease (6, 16, 28, 32, 40). Th2-like cytokine mRNA has been demonstrated in cells from lung tissue or bronchoalveolar lavage (BAL) specimens after RSV challenge of FI-RSV-immunized mice (17, 32, 40). In addition, in vivo studies using antibody (Ab) blockade showed that the enhanced histopathology in FI-RSV-immune mice challenged with live virus could be eliminated by using anti-interleukin-4 (IL-4) and anti-IL-10 Abs but not anti-IL-12 Abs (6). Recent evidence suggests that CD8⁺ T lymphocytes may be important in directing the type of inflammatory response to RSV in challenge of G glycoprotein-sensitized mice (21, 31).One aspect of the FI-RSV immune response that has not been well characterized is the cytotoxic T-lymphocyte (CTL) response. There is limited information on major histocompatibility complex (MHC) class I-restricted CTLs after FI-RSV immunization (29), while the information about the CTL response after live-RSV infection has been well documented. Several studies have shown class I-restricted CTLs to kill predominantly target cells expressing the M, N, or F RSV protein (5, 7, 9, 26, 29, 41). The role of CTLs in the immune response to RSV is well illustrated by in vivo depletion studies with BALB/c mice (8, 18, 30). These studies suggest that both CD4⁺ (class II) and CD8⁺ lymphocytes are important for clearing RSV and that both contribute to the inflammatory response associated with infection. A vaccinia virus construct expressing RSV membrane-associated, nonglycosylated protein M2 has been affiliated with short-term protection in the BALB/c mouse (7). This protein does not induce neutralizing Abs, and therefore, protection likely is mediated by CTLs. Passive transfer of CD8⁺ T lymphocytes has been associated with both clearance of the virus and enhanced histopathology (1).In this report, we describe studies of CTL precursor (CTLp) frequencies in both live-RSV-infected and FI-RSV-immunized mice for MHC class I- and class II-restricted target cells. These studies demonstrate clear differences in the CTLp response between RSV and FI-RSV immunizations and provide additional approaches to identifying potential FI-RSV-induced enhanced disease mechanisms.     

Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.     

紫球藻胞外多糖抗呼吸道合胞病毒(RSV)活性研究

采用体外细胞培养的方法,在Hela细胞系上检测了来自紫球藻的胞外多糖及其组分的抗呼吸道病毒(RSV)活性。发现紫球藻胞外多糖对呼吸道合胞病毒具有强烈的抑制活性,同时对宿主细胞的抑制作用很小。分离组分中的强带电性组分ESPSⅥ活性最高,其TI值达3125,为阳性对照药病毒唑的40余倍。     

A Novel Influenza Virus Hemagglutinin-Respiratory Syncytial Virus (RSV) Fusion Protein Subunit Vaccine against Influenza and RSV

Influenza A virus and respiratory syncytial virus (RSV) cause substantial morbidity and mortality afflicting the ends of the age spectrum during the autumn through winter months in the United States. The benefit of vaccination against RSV and influenza using a subunit vaccine to enhance immunity and neutralizing antibody was investigated. Influenza virus hemagglutinin (HA) and RSV fusion (F) protein were tested as vaccine components alone and in combination to explore the adjuvant properties of RSV F protein on HA immunity. Mice vaccinated with HA and F exhibited robust immunity that, when challenged, had reduced viral burden for both influenza and RSV. These studies show an enhancing and cross-protective benefit of F protein for anti-HA immunity.     

Differential Histopathology and Chemokine Gene Expression in Lung Tissues following Respiratory Syncytial Virus (RSV) Challenge of Formalin-Inactivated RSV- or BBG2Na-Immunized Mice

A BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 microg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1beta, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.     

Latitudinal Variations in Seasonal Activity of Influenza and Respiratory Syncytial Virus (RSV): A Global Comparative Review

Background

There is limited information on influenza and respiratory syncytial virus (RSV) seasonal patterns in tropical areas, although there is renewed interest in understanding the seasonal drivers of respiratory viruses.

Methods

We review geographic variations in seasonality of laboratory-confirmed influenza and RSV epidemics in 137 global locations based on literature review and electronic sources. We assessed peak timing and epidemic duration and explored their association with geography and study settings. We fitted time series model to weekly national data available from the WHO influenza surveillance system (FluNet) to further characterize seasonal parameters.

Results

Influenza and RSV activity consistently peaked during winter months in temperate locales, while there was greater diversity in the tropics. Several temperate locations experienced semi-annual influenza activity with peaks occurring in winter and summer. Semi-annual activity was relatively common in tropical areas of Southeast Asia for both viruses. Biennial cycles of RSV activity were identified in Northern Europe. Both viruses exhibited weak latitudinal gradients in the timing of epidemics by hemisphere, with peak timing occurring later in the calendar year with increasing latitude (P<0.03). Time series model applied to influenza data from 85 countries confirmed the presence of latitudinal gradients in timing, duration, seasonal amplitude, and between-year variability of epidemics. Overall, 80% of tropical locations experienced distinct RSV seasons lasting 6 months or less, while the percentage was 50% for influenza.

Conclusion

Our review combining literature and electronic data sources suggests that a large fraction of tropical locations experience focused seasons of respiratory virus activity in individual years. Information on seasonal patterns remains limited in large undersampled regions, included Africa and Central America. Future studies should attempt to link the observed latitudinal gradients in seasonality of viral epidemics with climatic and population factors, and explore regional differences in disease transmission dynamics and attack rates.     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants

Rationale

Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions.

Methods

Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 10⁷ FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses.

Results

Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log₁₀ FFU/mL), which was maintained until day 6 (4.83 Log₁₀ FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log₁₀ M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log₁₀ M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log₁₀ M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants.

Conclusion

This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants.