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PubMed2002

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells.

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

Animals↗
PubMed2002

NK T cells contribute to expansion of CD8(+) T cells and amplification of antiviral immune responses to respiratory syncytial virus.

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Valpha14(+) natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129xC57BL/6, C57BL/6, and BALB/c CD1d(-/-) mice. CD8(+) T lymphocytes were reduced in CD1d(-/-) mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-gamma) production. Transient activation of NK T cells in CD1d(+/+) mice by alpha-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-gamma production and efficient induction of CD8(+)-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of alpha-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or alpha-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8(+) T cells and amplification of antiviral responses to RSV.

Animals↗
PubMed2004

Characterization of recombinant respiratory syncytial viruses with the region responsible for type 2 T-cell responses and pulmonary eosinophilia deleted from the attachment (G) protein.

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpDeltaG150-222 or rA2cpDeltaG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37 degrees C, but pinpoint morphology was observed at 39 degrees C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log(10)-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpDeltaG150-222 or rA2cpDeltaG177-220. These findings are important for the development of attenuated RSV vaccines.

Animals↗
PubMed2004

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV.

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.

Animals↗
PubMed2004

trans-Complementation allows recovery of human respiratory syncytial viruses that are infectious but deficient in cell-to-cell transmission.

Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported. To generate a system with which to study the roles of the viral transmembrane glycoproteins, including F, in the HRSV life cycle, we generated a cell line expressing a heterologous viral glycoprotein for complementation of glycoprotein function in trans. We previously demonstrated that the baculovirus GP64 protein or a chimeric form of GP64 carrying the 12 C-terminal amino acids of the HRSV F protein (GP(64/F)) can efficiently mediate HRSV infectivity and improve its stability, when expressed from an engineered HRSV genome. Here, we report the development of a stably transfected Vero cell line (Vbac) constitutively expressing the GP(64/F) protein. From the Vbac cell line, viruses that lacked the SH and F open reading frames (ORFs) or the SH, G, and F ORFs could be recovered from cDNAs. These viruses, designated RSDeltaSH,F and RSDeltaSH,G,F, respectively, had place-keeper ORFs inserted in place of the deleted ORFs to maintain authentic transcription levels. In the Vbac cell line, RSDeltaSH,f and RSDeltaSH,G,F could be amplified to near wild-type-level titers, and the resulting viruses were infectious to Vero and HEp-2 cells. After entry into Vero or HEp-2 cells, however, neither virus RSDeltaSH,F nor virus RSDeltaSH,G,F was able to spread in the infected cultures. Growth analyses showed that infectious virions were not produced in Vero or HEp-2 cells infected with RSDeltaSH,F and RSDeltaSH,G,F. Combined, these data provide direct evidence that HRSV F is an essential viral protein required for cell-to-cell transmission and demonstrate that complementation with the GP64 protein in trans constitutes a powerful tool for the study of the role of individual HRSV transmembrane glycoproteins in virus assembly, morphogenesis, and pathogenesis. In addition, the ability to generate infectious but nonspreading viruses may provide an alternative approach for the development of safe and stable HRSV vaccine candidates.

Animals↗
PubMed2004

Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys.

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.

Animals↗
PubMed2005

Modulation of protective immunity, eosinophilia, and cytokine responses by selective mutagenesis of a recombinant G protein vaccine against respiratory syncytial virus.

Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128 to 229) of the respiratory syncytial virus (RSV) (Long strain) G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids to vaccine efficacy. Mice were immunized with a total of 10 wild-type or mutant Trx-G proteins and challenged intranasally with RSV. Striking differences in the induction of RSV G-protein-specific antibodies, protection against RSV challenge, cytokine RNA responses, and induction of RSV-associated eosinophilic inflammation were observed among the mutant proteins examined. Taken together, the results identify a critical role for specific amino acids in the induction of protective immunity and priming for eosinophilia against RSV.

Adjuvants, Immunologic↗
PubMed2006

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector.

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-alpha/beta) 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-alpha/beta receptor.

Animals↗
PubMed2005

Protection against bovine respiratory syncytial virus challenge following a single dose of vaccine in young calves with maternal antibody.

Twenty-one young calves with maternally derived antibody to bovine respiratory syncytial virus (BRSV) were divided into three groups of seven, each group balanced for BRSV antibody titre. The calves had no evidence of previous exposure to BRSV. The calves in one group were given a single dose of a monovalent modified live BRSV vaccine; the calves in the second group were given a single dose of an inactivated combined BRSV, parainfluenza virus type 3, Mannheimia haemolytica vaccine and the calves in the third group were left as unvaccinated controls. Three weeks after the single doses of vaccine, all the calves were challenged with BRSV. The clinical signs of disease were mild, and virus excretion was limited to two calves in the group given the inactivated vaccine, compared with six in the negative controls (P = 0.05) and five in the group given the live vaccine. The mean virus excretion titres after the challenge were not significantly different between the groups. There was little seroconversion before the challenge, but six of the seven calves in the group given the inactivated vaccine showed significant seroconversion within two weeks after the challenge, compared with only one calf in each of the other two groups (P = 0.015).

Animals↗
PubMed2001

Validation of synthetic peptide enzyme immunoassays in differentiating two subgroups of ruminant respiratory syncytial virus.

Subgroup-specific peptide-based enzyme immunoassays from each respective G-glycoprotein of the ovine and the bovine respiratory syncytial virus (RSV) were developed to detect RSV-specific IgG responses in cattle. Antigenic peptides from the respective G-glycoprotein were identified from the extracellular central hydrophobic region (amino acids 158-189) located between 2 mucin-rich regions. These antigenic peptides identified by epitope mapping from each G-glycoprotein were synthesized and used to develop the subgroup-specific enzyme immunoassays. The negative cutoff for each enzyme immunoassay was established as the mean optical density of indirect immunofluorescent antibody-negative bovine sera plus 3 SDs. The sensitivity (82.9%) and specificity (100%) of the bovine enzyme immunoassay and the specificity (95.8%) of the ovine enzyme immunoassay were determined by comparison with indirect immunofluorescence (used as the "gold standard"). The negative and positive predictive values were calculated for each assay. The presence of serum antibody to ovine RSV in cattle implies that this virus infects cattle and may contribute to the pathogenesis of bovine respiratory disease.

Animals↗
PubMed2001

Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay.

Subgroup-specific peptide-based enzyme-linked immunosorbent assays from the G-protein of the ovine and bovine respiratory syncytial virus (RSV), respectively, were used to determine the prevalence of the ovine and bovine subgroup strains of RSV infections in cattle. A total of 1,102 bovine serum samples were obtained from 6 diagnostic laboratories located in the northwestern and the southeastern USA and were tested for antibody to either the bovine or ovine subgroups of RSV. Antibody to viruses from each subgroup was present in samples from each region and all states tested. The Southeast had a higher prevalence of the bovine subgroup strains (69.5%). Then did the Northwest (40.9%). The prevalence of the ovine strain was similar for the two regions (16.7% in the southeast, 14.9% in the northwest). The overall prevalence was 56.6% for the bovine strain and 15.9% for the ovine strain. These results suggest members of the ovine subgroup of RSV circulate in the cattle population but with less frequency than those viruses of the bovine subgroup.

Animals↗
PubMed2005

B-cell depletion inhibits arthritis in a collagen-induced arthritis (CIA) model, but does not adversely affect humoral responses in a respiratory syncytial virus (RSV) vaccination model.

We report the development of a mouse B cell-depleting immunoconjugate (anti-CD22 monoclonal antibody [mAb] conjugated to calicheamicin) and its in vivo use to characterize the kinetics of CD22+ B-cell depletion and reconstitution in murine primary and secondary lymphoid tissues. The effect of B-cell depletion was further studied in a murine collagen-induced arthritis (CIA) model and a respiratory syncytial virus (RSV) vaccination model. Our results show that (1) the immunoconjugate has B-cell-specific in vitro and in vivo cytotoxicity; (2) B-cell reconstitution starts in the bone marrow and spleen around day 30 after depletion and is completed in all tissues tested by day 50; (3) B-cell depletion inhibits the development of clinical and histologic arthritis in the CIA model; (4) depletion of type II collagen antibody levels is not necessary for clinical and histologic prevention of CIA; and (5) B-cell depletion does not adversely affect memory antibody responses after challenge nor clearance of infectious virus from lungs in the RSV vaccination model. These results demonstrate for the first time that only B-cell reduction but not type II collagen antibody levels correlate with the prevention of arthritis and represent key insights into the role of CD22-targeted B-cell depletion in mouse autoimmunity and vaccination models.

Animals↗
PubMed2001

Bovine respiratory syncytial virus (BRSV) pneumonia in beef calf herds despite vaccination.

The present report describes the clinical, pathological, serological and virological findings in calves from 2 larger Danish beef herds experiencing outbreaks of pneumonia. The calves had been vaccinated with an inactivated bovine respiratory syncytial virus (BRSV) vaccine 2 months prior to the outbreak. The clinical signs comprised nasal discharge, pyrexia, cough and increased respiratory rates. A total of 28 calves died in the 2 herds. The laboratory investigations revealed that BRSV was involved and probably initiated both outbreaks. Furthermore, the serological results suggested that the vaccine induced only sparse levels of antibodies probably due to the presence of maternally derived antibodies at the time of vaccination. Necropsy findings in 5 calves revealed changes typical for infectious pneumonia with involvement of BRSV. In conclusion, vaccination of calves against BRSV in 2 Danish beef herds failed to protect the calves against severe or even fatal BRSV mediated respiratory disease 2 months later.

Animals↗
PubMed2005

Respiratory syncytial virus infections: characteristics and treatment.

In this review, we describe the history, epidemiology and clinical manifestations of infections attributed to respiratory syncytial virus (RSV) in children. At present, no cure exists for RSV infection but commonly employed palliative treatments include oxygen and inhaled beta(2)-adrenoceptor agonists, such as salbutamol, to relieve the wheezing and increased bronchiolar smooth muscle constriction. Adrenaline (epinephrine) has been found to be superior to the selective beta(2)-adrenoceptor agonists. Oral or inhaled corticosteroids should counteract the inflammatory response to RSV infection but their effectiveness is controversial. Inhaled ribavirin is the only licensed antiviral product approved for the treatment of RSV lower respiratory-tract infection in hospitalized children, although its use is now restricted to high-risk infants. Other treatments considered are nasopharyngeal suctioning, surfactant therapy, recombinant human deoxyribonuclease I, heliox (helium:oxygen) and inhaled nitric oxide. Prevention of infection by RSV antibodies is another strategy and, currently, palivizumab is the only safe, effective and convenient preventative treatment for RSV disease in high-risk populations of infants and young children. Its cost-effectiveness, however, has been questioned. Both live attenuated and subunit vaccines against RSV infection have been developed but so far there is no safe and effective vaccine available. Finding effective treatments and prophylactic measures remains a major challenge for the future.

Anti-Inflammatory Agents↗
PubMed2005

New generation live vaccines against human respiratory syncytial virus designed by reverse genetics.

Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non-temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN alpha/beta antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use.

Animals↗
PubMed2005

Carriers for the delivery of a vaccine against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in young children and the elderly. Despite its clinical importance, there is no licensed vaccine available at present. Vaccine development has been hampered by observations of increased pathology after RSV infection in infants vaccinated with formalin-inactivated RSV; incomplete immunity following natural infection; and the need to be effective during the neonatal period when levels of maternal antibody are high. Four categories of RSV vaccine carriers--live-attenuated RSVs, recombinant vectors expressing the protective antigens of RSV, DNA vaccines and subunit vaccines--have been evaluated in animal models and/or clinical trials. So far, studies with live-attenuated virus vaccines highlight the need to improve immunogenicity whilst maintaining a suitable level of attenuation. Studies with recombinant vectors, DNA and subunit vaccines illustrate the pivotal nature of the vaccine carrier in determining the balance between immune-mediated protection against infection and the induction of immune-mediated pulmonary pathology.

Animals↗
PubMed2004

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves.

OBJECTIVE: To determine whether a single intranasal dose of modified-live bovine respiratory syncytial virus (BRSV) vaccine protects calves from BRSV challenge and characterize cell-mediated immune response in calves following BRSV challenge. ANIMALS: 13 conventionally reared 4- to 6-week-old Holstein calves. PROCEDURES: Calves received intranasal vaccination with modified live BRSV vaccine (VC-group calves; n = 4) or mock vaccine (MC-group calves; 6) 1 month before BRSV challenge; unvaccinated control-group calves (n = 3) underwent mock challenge. Serum virus neutralizing (VN) antibodies were measured on days -30, -14, 0, and 7 relative to BRSV challenge nasal swab specimens were collected for virus isolation on days 0 to 7. At necropsy examination on day 7, tissue specimens were collected for measurement of BRSV-specific interferon gamma (IFN-gamma) production. Tissue distribution of CD3+ T and BLA.36+ B cells was evaluated by use of immunohistochemistry. RESULTS: The MC-group calves had significantly higher rectal temperatures, respiratory rates, and clinical scores on days 5 to 7 after BRSV challenge than VC-group calves. No difference was seen between distributions of BRSV in lung tissue of VC- and MC-group calves. Production of BRSV-specific IFN-gamma was increased in tissue specimens from VC-group calves, compared with MC- and control-group calves. Virus-specific IFN-gamma production was highest in the mediastinal lymph node of VC-group calves. Increased numbers of T cells were found in expanded bronchial-associated lymphoid tissue and airway epithelium of VC-group calves. CONCLUSIONS AND CLINICAL RELEVANCE: An intranasal dose of modified-live BRSV vaccine can protect calves against virulent BRSV challenge 1 month later.

Administration, Intranasal↗