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.     

Recombinant human respiratory syncytial virus (RSV) from cDNA and construction of subgroup A and B chimeric RSV

OBJECTIVE: The present study was performed to discriminate between central and peripheral effects of noradrenaline (NA) in normotensive, non-obese, type 2 diabetic patients. METHODS: Study I: In 10 patients and 10 healthy volunteer (HV) cumulative doses of NA were infused intravenously until mean arterial pressure (MAP) rose with 20 mmHg, and subsequently the effects on the forearm blood flow (FBF) was measured. Also, the FBF response to intra-arterial NA (0.025, 0.1, 0.4 micrograms min-1) was measured. Study II: In 13 patients and 14 HV the venous constrictor response to a cumulative local infusion of NA in a dorsal hand vein was determined. RESULTS: In study I the circulating plasma NA concentrations inducing a rise in MAP of 20 mmHg, were lower in the type 2 patients relative to the HV (p < 0.01). The relationship between changes in pressure and changes in heart rate were similar in both groups. Moreover, FBF responsiveness to intra-arterial NA was not different between the two groups. The slopes of the delta MAP/NA regression lines were correlated with basal insulin levels and relative insulin resistance in the healthy volunteers (R = 0.77, p < 0.01, and R = 0.83, p < 0.01), but not in the type 2 diabetic patients. In study II no differences were observed in the dose generating half maximum (ED50) and the maximum (Emax) response to NA between the type 2 patients and the HV. CONCLUSIONS: Non-obese normotensive type 2 patients have an increased pressor response to NA, which is not based upon a defect in skeletal muscle resistance arterioles, peripheral veins, or a defect in the baroreceptor system. Therefore, in type 2 diabetes the noradrenergic responsiveness of other vascular beds, such as the splanchnic or renal, must be enhanced.     

A model for respiratory syncytial virus (RSV) infection based on experimental aerosol exposure with bovine RSV in calves

Five conventionally kept calves aged between 17 and 24 days were experimentally infected with bovine respiratory syncytial virus (BRSV) by aerosol in order to mimic the natural infection route. The calves were killed and autopsies performed 7 days after the first virus challenge. The BRSV isolate used induced tracheitis, bronchitis and atelectasis in infected calves. The only virus which could be isolated from the lungs of the calves was BRSV. In addition, Mycoplasma bovirhinis was isolated from the lungs or/and trachea of two calves. The clinical and histopathological findings, as well as the detection of BRSV antigens by immunofluorescence in the epithelial cells of lung and trachea, and the reisolation of the virus from bronchoalveolar lavage fluids of all inoculated calves, provided confirmation of successful infection with BRSV.     

Respiratory syncytial virus (RSV) disease and prospects for its control

Respiratory syncytial virus (RSV) is a major virus pathogen of infants and young children, an important cause of disease in adults and is responsible for a significant amount of excess morbidity and mortality in the elderly. It also can be devastating in immunosuppressed populations. Vaccines are being developed, but none are currently licensed. Moreover, even if one or more are approved, they may not be suitable for some populations vulnerable to RSV (e.g. very young infants and the immunosuppressed). Ribavirin and immunoglobulin preparations with high titers of RSV-specific neutralizing antibodies are currently approved for use to treat and prevent RSV infection. However, neither of these is cost-effective or simple to administer. New agents are needed to reduce the impact of RSV. This review is concerned with the means currently available for controlling RSV, the search for new agents effective against this virus, and future prospects for preventing and treating RSV infections.     

Priming with secreted glycoprotein G of respiratory syncytial virus (RSV) augments interleukin-5 production and tissue eosinophilia after RSV challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4+ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.     

Increased number of T cells committed to IL-5 production after respiratory syncytial virus (RSV) infection of human mononuclear cells in vitro

We examined changes in the cytokine profile of T cells induced by in vitro infection with RSV. Isolated mononuclear cells from 27 healthy adults and six infants were infected with RSV at a concentration of 3 MOI (multiplicity of infection). After 48 h cells were restimulated with phorbol ester and ionomycin in the presence of monensin for 5 h. The intracellular expression of viral antigen, the cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-gamma), and the expression of surface markers were assessed by immunofluorescent staining and flow cytometry. There was a significant (P<0.001) rise of IL-5 expression in RSV-infected cultures in comparison with uninfected cultures from the same individuals, whereas there were no changes in the expression of the other lymphokines. The increase in IL-5 generation depended on viable infectious RSV rather than inactivated virus. RSV infection as well as IL-5 production in T cells were confined to the CD8 subpopulation. However, there was no simultaneous expression of RSV antigen and IL-5. Purified T cells did not show any increase in IL-5 generation. However, when the rate of RSV infection was enhanced in monocytes by means of a specific monoclonal antibody, co-cultured T cells displayed an increase of IL-5 production compared with samples with ordinary low rate RSV infection. It is therefore likely that the increased commitment of lymphocytes to produce IL-5 after RSV infection in vitro is mediated by monocytes or other antigen-presenting cells.     

A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

A critical issue has been the observation that vaccination of children with a formalin-inactivated respiratory syncytial virus (RSV) vaccine is associated with disease enhancement. We have taken advantage of bovine RSV and our experience with this disease in calves to develop a natural model that parallels human disease. Using formalin-inactivated bovine RSV vaccine calves were either sham-vaccinated/infected, vaccinated/infected, or vaccinated/sham-infected and their clinical signs, pulmonary function, and histological lung lesions quantitatively scored. Interestingly there was significantly greater disease in vaccinated/infected calves and histological lesions in calves were similar to those of affected children. Finally, we note that vaccination did not induce neutralizing antibodies, but IgG antibodies were detected by ELISA. Our model of RSV enhanced disease is important because it provides quantifiable evidence of disease severity that can be applied to evaluate the mechanisms of immunopathology and the safety of candidate RSV vaccines.     

Immune response to baculovirus expressed protein fragment amino acids 190-289 of respiratory syncytial virus (RSV) fusion protein

At least two neutralizing epitopes have been identified in the amino acid (aa) sequence 190-289 of the RSV fusion protein. The authors expressed this region in insect cells (bF190-289) and compared the immune response to bF190-289 with that induced by baculovirus expressed full-length fusion protein (bF). As with bF, mice primed with bF190-289 produced exclusively antibodies of IgG1 isotype, generated neutralizing antibodies, reduced significantly the virus titer (about a half log10 reduction) after RSV challenge and induced a Helper T (Th) 2 cell response in mediastinal lymph node cells (MLNC) restimulated in vitro. Thus, the aa sequence 190-289 represents a major immunogenic region of the RSV fusion protein.     

Antigenic and genomic diversity of central European respiratory syncytial virus strains

Thirty-two RSV strains recovered during the winter months of 1987/88 to 1993/94 from hospitalized children in Vienna, Austria and Zagreb, Croatia were analysed for antigenic and genetic variations. Twenty-nine of the 32 isolates investigated belonged to group A and 3 to group B, with the majority of infections caused by subgroup A1 (21 of 29). Restriction endonuclease mapping of PCR products derived from parts of the N and G gene of 18 group A strains identified 3 distinct lineages, very similar to those defined by analysis of recurrent epidemics in Birmingham, United Kingdom during the same period. Results of this study provide further information on the global pattern of RSV and show that very similar viruses are present simultaneously in widely separated areas.     

A live human parainfluenza type 3 virus vaccine is attenuated and immunogenic in healthy infants and children

The safety, infectivity, immunogenicity, and phenotypic stability of the cold-passaged (cp) candidate vaccine cp-45, a cold-adapted (ca), temperature-sensitive (ts) mutant of the JS strain of human parainfluenza virus type 3 (HPIV-3), was evaluated in 114 children 6 months to 10 years old in a randomized, placebo-controlled, double-blind trial. The cp-45 vaccine was well tolerated when given intranasally to parainfluenza virus type 3 (PIV-3)-seropositive and -seronegative children. With 10(4) or 10(5) TCID50 of cp-45 vaccine, 86% of seronegative vaccines were infected, 83% of whom shed virus at a mean peak titer of 10(22) pfu/mL. Virus present in respiratory specimens retained the ts phenotype, and each of 86 PIV-3 isolates tested retained both the ca and ts phenotypes. One dose of 10(5) TCID50 of vaccine induced a serum hemagglutination-inhibiting antibody response in 81% of vaccinees; the geometric mean titer was 1:32. These studies indicate that the cp-45 HPIV-3 vaccine is satisfactorily attenuated, infectious, immunogenic, and phenotypically stable and merits further evaluation in infants and young children.     

Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in the institutionalized elderly

The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in an open label study in 37 frail institutionalized persons over age 65. Vaccination was well tolerated without significant side-effects. Thirty-six of 37 volunteers completed the study. Nineteen of 36 (53%) vaccinees had a greater than or equal to fourfold increase in IgG to F protein at 4 weeks and 17 (47%) had a greater than or equal to fourfold rise in neutralizing titers to either group A or B virus. Although response rate to PFP-2 vaccine in the frail elderly was somewhat diminished compared to results in the healthy elderly, the vaccine was well tolerated and relatively immunogenic.     

Antigenic structure of human respiratory syncytial virus fusion glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

Immune responses of infants to infection with respiratory viruses and live attenuated respiratory virus candidate vaccines

Respiratory viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIV), and the influenza viruses cause severe lower respiratory tract diseases in infants and children throughout the world. Experimental live attenuated vaccines for each of these viruses are being developed for intranasal administration in the first weeks or months of life. A variety of promising RSV, PIV-3, and influenza virus vaccine strains have been developed by classical biological methods, evaluated extensively in preclinical and clinical studies, and shown to be attenuated and genetically stable. The ongoing clinical evaluation of these vaccine candidates, coupled with recent major advances in the ability to develop genetically engineered viruses with specified mutations, may allow the rapid development of respiratory virus strains that possess ideal levels of replicative capacity and genetic stability in vivo. A major remaining obstacle to successful immunization of infants against respiratory virus associated disease may be the relatively poor immune response of very young infants to primary virus infection. This paper reviews the immune correlates of protection against disease caused by these viruses, immune responses of infants to naturally-acquired infection, and immune responses of infants to experimental infection with candidate vaccine viruses.     

Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60

The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in a randomized placebo-controlled, double-blind study of 64 healthy adults over age 60. Vaccination was well tolerated with no significant acute side-effects. Twenty-nine of 33 vaccinees (87%) showed a greater than or equal to fourfold rise in serum IgG to the F protein of RSV at 8 weeks post vaccination. Twenty of 33 vaccine recipients (61%) had a greater than or equal to fourfold rise in serum neutralizing titer to group A and/or group B RSV. Response to vaccination was inversely correlated with pre-immunization serum neutralizing titers. Active surveillance throughout the ensuring winter identified three RSV infections in the placebo group and none in the vaccine group. Thus, PFP-2 was found to be safe and immunogenic in healthy older adults.     

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies

Infections with respiratory syncytial virus (RSV) are characterized by frequently occurring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.