Respiratory syncytial virus induced type I IFN production by pDC is regulated by RSV-infected airway epithelial cells, RSV-exposed monocytes and virus specific antibodies.

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14(+) myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,ß receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.

Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice.

Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn(-/-) mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4(+) T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8(+) T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.

The comparative genomics of human respiratory syncytial virus subgroups A and B: genetic variability and molecular evolutionary dynamics.

Genomic variation and related evolutionary dynamics of human respiratory syncytial virus (RSV), a common causative agent of severe lower respiratory tract infections, may affect its transmission behavior. RSV evolutionary patterns are likely to be influenced by a precarious interplay between selection favoring variants with higher replicative fitness and variants that evade host immune responses. Studying RSV genetic variation can reveal both the genes and the individual codons within these genes that are most crucial for RSV survival. In this study, we conducted genetic diversity and evolutionary rate analyses on 36 RSV subgroup B (RSV-B) whole-genome sequences. The attachment protein, G, was the most variable protein; accordingly, the G gene had a higher substitution rate than other RSV-B genes. Overall, less genetic variability was found among the available RSV-B genome sequences than among RSV-A genome sequences in a comparable sample. The mean substitution rates of the two subgroups were, however, similar (for subgroup A, 6.47 × 10(-4) substitutions/site/year [95% credible interval {CI 95%}, 5.56 × 10(-4) to 7.38 × 10(-4)]; for subgroup B, 7.76 × 10(-4) substitutions/site/year [CI 95%, 6.89 × 10(-4) to 8.58 × 10(-4)]), with the time to their most recent common ancestors (TMRCAs) being much lower for RSV-B (19 years) than for RSV-A (46.8 years). The more recent RSV-B TMRCA is apparently the result of a genetic bottleneck that, over longer time scales, is still compatible with neutral population dynamics. Whereas the immunogenic G protein seems to require high substitution rates to ensure immune evasion, strong purifying selection in conserved proteins such as the fusion protein and nucleocapsid protein is likely essential to preserve RSV viability.

Genetic variability among complete human respiratory syncytial virus subgroup A genomes: bridging molecular evolutionary dynamics and epidemiology.

Human respiratory syncytial virus (RSV) is an important cause of severe lower respiratory tract infections in infants and the elderly. In the vast majority of cases, however, RSV infections run mild and symptoms resemble those of a common cold. The immunological, clinical, and epidemiological profile of severe RSV infections suggests a disease caused by a virus with typical seasonal transmission behavior, lacking clear-cut virulence factors, but instead causing disease by modifying the host's immune response in a way that stimulates pathogenesis. Yet, the interplay between RSV-evoked immune responses and epidemic behavior, and how this affects the genomic evolutionary dynamics of the virus, remains poorly understood. Here, we present a comprehensive collection of 33 novel RSV subgroup A genomes from strains sampled over the last decade, and provide the first measurement of RSV-A genomic diversity through time in a phylodynamic framework. In addition, we map amino acid substitutions per protein to determine mutational hotspots in specific domains. Using Bayesian genealogical inference, we estimated the genomic evolutionary rate to be 6.47 × 10(-4) (credible interval: 5.56 × 10(-4), 7.38 × 10(-4)) substitutions/site/year, considerably slower than previous estimates based on G gene sequences only. The G gene is however marked by elevated substitution rates compared to other RSV genes, which can be attributed to relaxed selective constraints. In line with this, site-specific selection analyses identify the G gene as the major target of diversifying selection. Importantly, statistical analysis demonstrates that the immune driven positive selection does not leave a measurable imprint on the genome phylogeny, implying that RSV lineage replacement mainly follows nonselective epidemiological processes. The roughly 50 years of RSV-A genomic evolution are characterized by a constant population size through time and general co-circulation of lineages over many epidemic seasons - a conclusion that might be taken into account when developing future therapeutic and preventive strategies.

Specific dietary oligosaccharides increase Th1 responses in a mouse respiratory syncytial virus infection model.

Breast feeding reduces the risk of developing severe respiratory syncytial virus (RSV) infections in infants. In addition to maternal antibodies, other immune-modulating factors in human milk contribute to this protection. Specific dietary prebiotic oligosaccharides, similar to oligosaccharides present in human milk, were evaluated in a C57BL/6 mouse RSV infection model. During primary RSV infection, increased numbers of RSV-specific CD4(+) T cells producing gamma interferon (IFN-γ) were found in the lungs at days 8 to 10 postinfection in mice receiving diet containing short-chain galactooligosacharides, long-chain fructooligosaccharides, and pectin-derived acidic oligosaccharides (termed scGOS/lcFOS/pAOS). In a Th2-skewed formalin-inactivated (FI)-RSV vaccination model, the prebiotic diet reduced RSV-specific Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13)-producing CD4(+) T cells in the lung and the magnitude of airway eosinophilia at day 4 and 6 after infection. This was accompanied by a decreased influx of inflammatory dendritic cells (CD11b(+)/CD11c(+)) and increased numbers of IFN-γ-producing CD4(+) and CD8(+) T cells at day 8 after viral challenge. These findings suggest that specific dietary oligosaccharides can influence trafficking and/or effector functions of innate immune, CD4(+), and CD8(+) T cell subsets in the lungs of RSV-infected mice. In our models, scGOS/lcFOS/pAOS had no effect on weight but increased viral clearance in FI-RSV-vaccinated mice 8 days after infection. The increased systemic Th1 responses potentiated by scGOS/lcFOS/pAOS might contribute to an accelerated Th1/Th2 shift of the neonatal immune system, which might favor protective immunity against viral infections with a high attack rate in early infancy, such as RSV.

Influencing mucosal homeostasis and immune responsiveness: the impact of nutrition and pharmaceuticals.

Both nutrition and orally ingested drugs pass the gastrointestinal mucosa and may affect the balance between the mucosal immune system and microbial community herein, i.e. affecting composition of the microbial community as well as the status of local immune system that controls microbial composition and maintains mucosal integrity. Numerous ways are known by which the microbial community stimulates mammalian host's immune system and vice versa. The communication between microbiota and immune system is principally mediated by interaction of bacterial components with pattern recognition receptors expressed by intestinal epithelium and various local antigen-presenting cells, resulting in activation or modulation of both innate and adaptive immune responses. Current review describes some of the factors influencing development and maintenance of a proper mucosal/immune balance, with special attention to Toll like receptor signaling and regulatory T cell development. It further describes examples (antibiotic use, HIV and asthma will be discussed) showing that disruption of the balance can be linked to immune function failure. The therapeutic potential of nutritional pharmacology herein is the main focus of discussion.

RSV 2010: Recent advances in research on respiratory syncytial virus and other pneumoviruses.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are important causes of acute respiratory tract disease in infants, immunocompromised patients and the elderly. The Seventh International RSV symposium was held in Rotterdam, the Netherlands, from December 2-5, 2010. This symposium is the flagship event for leading investigators engaged in RSV and HMPV research around the world. The objective of the symposium was to provide a forum to review recent advances in research on RSV, HMPV and other pneumoviruses. More than 200 young and established investigators attended the meeting. Over a hundred papers were presented in 55 oral presentations and six poster sessions, providing all participants the opportunity to share and to discuss their work. The Chanock lecture, instituted in 2003 to acknowledge important contributors to RSV research, was presented by Peter Collins. As a preface to his lecture, he presented an in memoriam of the late Dr. Robert M. Chanock, who played a key role in the characterization of RSV as a human pathogen. The current report presents highlights of the meeting, covering topics from basic virology, pathogenesis and immunology to clinical studies, therapeutics and vaccine development.

Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV.

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4(+)/IFN-γ(+) T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8(+) T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology.

Serum antibodies critically affect virus-specific CD4+/CD8+ T cell balance during respiratory syncytial virus infections.

Following infection with respiratory syncytial virus (RSV), reinfection in healthy individuals is common and presumably due to ineffective memory T cell responses. In peripheral blood of healthy adults, a higher CD4(+)/CD8(+) memory T cell ratio was observed compared with the ratio of virus-specific effector CD4(+)/CD8(+) T cells that we had found in earlier work during primary RSV infections. In mice, we show that an enhanced ratio of RSV-specific neutralizing to nonneutralizing Abs profoundly enhanced the CD4(+) T cell response during RSV infection. Moreover, FcγRs and complement factor C1q contributed to this Ab-mediated enhancement. Therefore, the increase in CD4(+) memory T cell response likely occurs through enhanced endosomal Ag processing dependent on FcγRs. The resulting shift in memory T cell response was likely amplified by suppressed T cell proliferation caused by RSV infection of APCs, a route important for Ag presentation via MHC class I molecules leading to CD8(+) T cell activation. Decreasing memory CD8(+) T cell numbers could explain the inadequate immunity during repeated RSV infections. Understanding this interplay of Ab-mediated CD4(+) memory T cell response enhancement and infection mediated CD8(+) memory T cell suppression is likely critical for development of effective RSV vaccines.

Distinct abnormalities in the innate immune system of children with Down syndrome.

OBJECTIVE: To analyze the frequency and phenotype of cells of the innate immune system in the peripheral blood of children with Down syndrome (DS). STUDY DESIGN: Flow cytometric analysis of expression of cell surface markers was performed in children with DS (n = 41) and healthy age-matched controls (n = 41). RESULTS: Compared with controls, children with DS had significantly lower absolute total leukocyte counts, lymphocytes, monocytes, and granulocytes, but 1.5-times higher absolute numbers of CD14(dim)CD16(+) monocytes (147 x 10(6)/L vs 93 x 10(6)/L; P = .02). This difference is fully explained by a higher percentage of CD14(dim)CD16(+) monocytes within the monocyte compartment (28.7% vs 13.4%; P <.001). The absolute numbers of myeloid dendritic cells were lower in DS (13.8 x 10(6)/L vs 22.7 x 10(6)/L; P <.001). The numbers of plasmacytoid dendritic cells and natural killer cells were normal. Absolute numbers of invariant natural killer T cells were very low overall, but significantly lower in children with DS than in controls (1.2 x 10(6)/L vs 3.7 x 10(6)/L; P = .01). CONCLUSIONS: Children with DS exhibited distinct abnormalities in cells of the innate immune system. Most strikingly, they had a high number of proinflammatory CD14(dim)CD16(+) monocytes. This elevated level of CD14(dim)CD16(+) monocytes may play an important role in the onset and maintenance of chronic inflammatory disease in DS.

A systemic neutrophil response precedes robust CD8(+) T-cell activation during natural respiratory syncytial virus infection in infants.

Severe primary respiratory syncytial virus (RSV) infections are characterized by bronchiolitis accompanied by wheezing. Controversy exists as to whether infants suffer from virus-induced lung pathology or from excessive immune responses. Furthermore, detailed knowledge about the development of primary T-cell responses to viral infections in infants is lacking. We studied the dynamics of innate neutrophil and adaptive T-cell responses in peripheral blood in relation to the viral load and parameters of disease in infants admitted to the intensive care unit with severe RSV infection. Analysis of primary T-cell responses showed substantial CD8(+) T-cell activation, which peaked during convalescence. A strong neutrophil response, characterized by mobilization of bone marrow-derived neutrophil precursors, preceded the peak in T-cell activation. The kinetics of this neutrophil response followed the peak of clinical symptoms and the viral load with a 2- to 3-day delay. From the sequence of events, we conclude that CD8(+) T-cell responses, initiated during primary RSV infections, are unlikely to contribute to disease when it is most severe. The mobilization of precursor neutrophils might reflect the strong neutrophil influx into the airways, which is a characteristic feature during RSV infections and might be an integral pathogenic process in the disease.

Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)-positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.

The association between intrauterine inflammation and spontaneous vaginal delivery at term: a cross-sectional study.

BACKGROUND: Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor. METHODS/RESULTS: In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher (18 vs 4%, p = 0.02) and amniotic fluid concentration of IL-6 was higher (3.1 vs 0.37 ng/mL, p<0.001). Similar results were obtained for IL-8 (10.93 vs 0.96 ng/mL, p<0.001) and percentage of detectable TNF-alpha (50 vs 4%, p<0.001). CONCLUSIONS: This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines.

Respiratory syncytial virus-induced activation and migration of respiratory dendritic cells and subsequent antigen presentation in the lung-draining lymph node.

In the respiratory tract, different dendritic cell (DC) populations guard a tight balance between tolerance and immunity to infectious or harmless materials to which the airways are continuously exposed. For infectious and noninfectious antigens administered via different routes, different subsets of DC might contribute during the induction of T-cell tolerance and immunity. We studied the impact of primary respiratory syncytial virus (RSV) infection on respiratory DC composition in C57BL/6 mice. We also tracked the migration of respiratory DC to the lymph nodes and studied antigen presentation by lung-derived and lymph node-resident DC to CD4(+) and CD8(+) T cells. We observed a massive influx of mainly CD103(-) CD11b(high) CD11c(+) conventional DC (cDC) and plasmacytoid DC during the first 7 days of RSV infection, while CD103(+) CD11b(low) CD11c(+) cDC disappeared from the lung. The two major subsets of lung tissue DC, CD103(+) CD11b(low) CD11c(+) and CD103(-) CD11b(high) CD11c(+) cDC, both transported RSV RNA to the lung-draining lymph node. Furthermore, these lung-derived cDC subsets as well as resident LN DC, which did not contain viral RNA, displayed viral antigen by major histocompatibility complex class I and class II to CD8(+) and CD4(+) T cells. Taken together, our data indicate that during RSV infections, at least three DC subsets might be involved during the activation of lymph node-homing naïve and memory CD4(+) and CD8(+) T cells.

The role of Toll-like receptors in regulating the immune response against respiratory syncytial virus.

Toll-like receptors (TLRs) play a distinct role in battling respiratory syncytial virus (RSV) infections. However, due to a lack of representative animal models and several early controversies, the field is unclear. In this systematic review, we have elucidated conflicting results and outlined important factors that might affect study outcomes. We reviewed studies that used different doses/viral strains, performed virus propagation in different cell lines, or used different mice strains. The following firm conclusions can be drawn: multiple TLRs activate innate immunity upon RSV infection; TLR4 can influence TLR2 expression, suggesting that optimal induction of multiple signaling pathways is required to elicit protective, rather than deleterious innate immune responses following infection; in mice, TLR4, TLR2/-6, and TLR7 have immune-stimulating properties, while TLR3 activation occurs later and appears to downregulate immune responses; in humans, polymorphism studies have demonstrated an important role for TLR4-signaling; and activation of TLR-signaling leads to antiviral cytokine production, such as TNF-a and IFNs. Viral factors may block these pathways, thereby contributing to immune evasion and RSV survival. A better understanding of the complex interplay between TLRs and severe RSV infections might lead to efficient prophylactic and therapeutic treatments, as well as the development of adequate vaccines combined with TLR adjuvants.

Dynamics of human respiratory virus-specific CD8+ T cell responses in blood and airways during episodes of common cold.

We determined the dynamics of CD8(+) T cells specific for influenza virus and respiratory syncytial virus in blood and tracheostoma aspirates of children during the course of respiratory infections. We showed that during localized respiratory infections the ratio of activated effector CD8(+) T cells to resting memory/naive CD8(+) T cells in peripheral blood increased significantly. Furthermore, the number of effector/memory T cells specific for respiratory viruses declined in blood and increased in the airways, suggesting that these T cells redistributed from blood to airways. T cells specific for the infecting virus were present in the airways for longer periods at increased levels than nonspecifically recruited bystander T cells. After clearance of the infection, the ratio of resting memory and naive CD8(+) T cells normalized in peripheral blood and also memory T cell numbers specific for unrelated viruses that declined during the infection due to bystander recruitment were restored. Taken together, these results showed a significant systemic T cell response during relatively mild secondary infections and extensive dynamics of virus-specific and nonspecific Ag-experienced T cells.

CD8+ T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections.

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7-CD38+HLA-DR+Granzyme B+CD127- could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9-12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.

Identification of a CD4 T cell epitope in the pneumonia virus of mice glycoprotein and characterization of its role in protective immunity.

Pneumonia virus of mice (PVM) causes bronchiolitis and pneumonia in mice. Infection is associated with high levels of viral replication in the lungs and results in the functional inactivation of pulmonary virus-specific CD8 T cells. Due to its similarity to severe human respiratory syncytial virus (RSV) infection, PVM infection in mice has been proposed as an alternative RSV model. Here, we have delineated the minimal requirements for protective T cell immunity in the PVM model. Immunization with a CD8 T cell epitope from the PVM phosphoprotein P, combined with the ovalbumin (OVA) CD4 T cell epitope, did not confer protective immunity against lethal PVM challenge, suggesting a possible role of cognate CD4 T cell immunity. To determine the role of PVM-specific CD4 T cell responses, we mapped a PVM CD4 T cell epitope in the glycoprotein G, using a panel of overlapping peptides. Although immunization with this epitope provided some protection, solid protective immunity was only observed after immunization with a combination of the PVM-specific CD4 and CD8 T cell epitopes. Analysis of post-challenge T cell responses in immunized mice indicated that G-specific pulmonary CD4 T cells displayed a mixed Th1/Th2 phenotype, which was characterized by the presence of both IL-5 and IFN-gamma secreting cells, in the absence of overt pathology.

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.