CD40 ligand (CD154) enhances the Th1 and antibody responses to respiratory syncytial virus in the BALB/c mouse.
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CD40 ligand (CD40L) is a cell surface costimulatory molecule expressed mainly by activated T cells. CD40L is critically important for T-B cell and T cell-dendritic cell interactions. CD40L expression promotes Th1 cytokine responses to protein Ags and is responsible for Ig isotype switching in B cells. Respiratory syncytial virus (RSV) is an important pathogen of young children and the elderly, which causes bronchiolitis and pneumonia. Studies of mice infected with RSV suggest that a Th2 cytokine response may be responsible for enhanced pulmonary disease. To investigate the effect CD40L has on RSV immunity, mice were infected simultaneously with RSV and either an empty control adenovirus vector or one expressing CD40L or were coimmunized with plasmid DNA vectors expressing CD40L and RSV F and/or G proteins and subsequently challenged with RSV. The kinetics of the intracellular and secreted cytokine responses, the cytotoxic T lymphocyte precursor frequency, NO levels in lung lavage, rates of virus clearance, and anti-RSV Ab titers were determined. These studies show that coincident expression of CD40L enhances the Th1 (IL-2 and IFN-gamma) cytokine responses, increases the expression of TNF-alpha and NO, accelerates virus clearance, and increases the anti-F and anti-G Ab responses. These data suggest that CD40L may have the adjuvant properties needed to optimize the safety and efficacy of RSV vaccines. (+info)
Requirement of a novel upstream response element in respiratory syncytial virus-induced IL-8 gene expression.
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Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. In this study, we compare mechanisms for induction of the CXC chemokine IL-8, in human type II alveolar (A549) cells by RSV infection and by stimulation with the cytokine TNF. Promoter deletion and mutagenesis experiments indicate that although the region from -99 to -54 nt is sufficient for TNF-induced IL-8 transcription, this region alone is not sufficient for RSV-induced IL-8 transcription. Instead, RSV requires participation of a previously unrecognized element, spanning from -162 to -132 nt, that we term the RSV response element (RSVRE), and a previously characterized element at -132 to -99 nt, containing an AP-1 binding site. RSV infection of A549 cells induces increased RSVRE- and AP-1-binding activities and increased synthesis of IFN regulatory factor-1 protein, which is present in the RSVRE-binding complex. These data confirm that the IL-8 gene enhancers are controlled in a stimulus-specific fashion and participation of distinct promoter elements is required to activate gene transcription. These observations are important for rational design of inhibitors of RSV-induced lung inflammation. (+info)
Serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus.
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Subunit vaccines formulated with purified fusion proteins from the A2 (PFP-2) or attenuated 248/404 (PFP-3) strains of respiratory syncytial virus (RSV) were evaluated, either alone or in combination with native attachment (G) protein, for their ability to augment serum neutralizing antibody titers in seropositive chimpanzees. The results suggested that combination vaccines enhanced serum neutralizing antibody titers against both laboratory strains and clinical isolates of RSV. When compared with PFP-2 alone, the resultant neutralizing antibody titers after vaccination with PFP-2+Ga protein were significantly elevated against 71% of A strains tested. In a confirmatory experiment, immunization with PFP-3+Ga+Gb proteins resulted in elevated serum neutralizing antibody titers against 86% of A and 50% of B strains tested versus injection with PFP-3 alone. The results suggest that subunit vaccines composed of both PFP and G proteins have more potential than PFP alone to augment neutralizing antibody titers in seropositive recipients. (+info)
Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism.
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Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF-2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination. (+info)
Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo.
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Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: DeltaNS1NS2, DeltaM2-2SH, DeltaM2-2NS2, DeltaSHNS1, DeltaSHNS2, and DeltaSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, DeltaM2-2NS2 was most attenuated. DeltaM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log(10) compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication. (+info)
Hospitalisation for RSV infection in ex-preterm infants-implications for use of RSV immune globulin.
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BACKGROUND: Respiratory syncytial virus (RSV) specific immune globulin is now being marketed for prevention of RSV infection in ex-preterm infants. However, there are no published UK data on the morbidity or mortality from RSV in these infants. AIMS: To determine the morbidity and mortality from RSV infection in a cohort of infants previously treated at a regional neonatal unit, and compare the cost of hospitalisation for RSV with the potential cost of administering RSV immune globulin (RSV-IG) prophylaxis. METHODS: Infants born at a gestation of less than 32 weeks were studied. Details of admissions for respiratory illness in the first two years of life were collected from hospital records, referring hospitals, and general practitioners. RESULTS: Data on 82 infants were collected. Up to three RSV seasons were encountered. The hospitalisation rate for confirmed RSV infection for the first season encountered was 4%. Rates of ward and paediatric intensive care unit admission were higher for infants with chronic lung disease. There were no deaths from RSV. RSV-IG would not have been cost effective for most infants. CONCLUSION: The morbidity and mortality rates from RSV observed in this group do not support the widespread introduction of RSV-IG prophylaxis for ex-preterm infants. (+info)
Characterization of a novel respiratory syncytial virus-specific human cytotoxic T-lymphocyte epitope.
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Respiratory syncytial virus (RSV) infection is a major cause of morbidity in childhood worldwide. The first human RSV-specific cytotoxic T-lymphocyte epitope to be defined is described. This HLA B7-restricted epitope in nucleoprotein (NP) was detectable in four healthy, B7-positive adult subjects using B7-RSV-NP tetrameric complexes to stain CD8(+) T cells. (+info)
Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry.
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Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type-2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV-infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL-4 and interferon-gamma (IFN-gamma) were measured by flow cytometry. The cells from RSV-infected infants produced more IL-4 and less IFN-gamma than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-gamma production increased with disease severity. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells. (+info)