Live Respiratory Syncytial Virus Research Articles

Rapid recovery in mice after combined nasal/oral immunization with killed respiratory syncytial virus

Based on the concept of a common mucosal immune system, the murine gastrointestinal tract was inoculated (oral) with three doses (5, 20, and 40 micrograms) of UV-inactivated respiratory syncytial virus (RSV) in order to elicit a virus-specific immune response in the respiratory tract. Only the 40 micrograms dose induced significant (P less than 0.01) anti-RSV-IgG rises in serum and lung wash compared to controls. To improve the immune response, mice were immunized intranasally under light anesthesia with the same 40 micrograms dosage regimen of killed RSV so that each dose passed through the nose and was swallowed. This combined nasal/oral immunization stimulated anti-RSV-IgG in serum, lung wash and nasal wash (P less than 0.001) and anti-RSV-IgA in lung and nasal wash (P less than 0.001) that were comparable to levels after infection with live RSV. Three days after challenge with live RSV, mice given combined nasal/oral immunization showed suppressed nasal virus shedding (P = 0.025). Nasal virus shedding correlated inversely with concentrations of anti-RSV-Ig in nasal secretions but did not correlate with concentrations in serum.

Cytolytic T-lymphocyte responses to respiratory syncytial virus: effector cell phenotype and target proteins

Cytolytic T-lymphocyte (CTL) activity specific for respiratory syncytial (RS) virus was investigated after intranasal infection of mice with RS virus, after intraperitoneal infection of mice with a recombinant vaccinia virus expressing the F glycoprotein, and after intramuscular vaccination of mice with Formalin-inactivated RS virus or a chimeric glycoprotein, FG, expressed from a recombinant baculovirus. Spleen cell cultures from mice previously infected with live RS virus or the F-protein recombinant vaccinia virus had significant CTL activity after one cycle of in vitro restimulation with RS virus, and lytic activity was derived from a major histocompatibility complex-restricted, Lyt2.2+ (CD8+) subset. CTL activity was not restimulated in spleen cells from mice that received either the Formalin-inactivated RS virus or the purified glycoprotein, FG. The protein target structures for recognition by murine CD8+ CTL were identified by using target cells infected with recombinant vaccinia viruses that individually express seven structural proteins of RS virus. Quantitation of cytolytic activity against cells expressing each target structure suggested that 22K was the major target protein for CD8+ CTL, equivalent to recognition of cells infected with RS virus, followed by intermediate recognition of F or N, slight recognition of P, and no recognition of G, SH, or M. Repeated stimulation of murine CTL with RS virus resulted in outgrowth of CD4+ CTL which, over time, became the exclusive subset in culture. Murine CD4+ CTL were highly cytolytic for RS virus-infected cells, but they did not recognize target cells infected with any of the recombinant vaccinia viruses expressing the seven RS virus structural proteins. Finally, the CTL response in peripheral blood mononuclear cells of adult human volunteers was investigated. The detection of significant levels of RS virus-specific cytolytic activity in these cells was dependent on at least two restimulations with RS virus in vitro, and cytolytic activity was derived primarily from the CD4+ subset.

Mechanism of lung injury in cotton rats immunized with formalin-inactivated respiratory syncytial virus.

Respiratory syncytial virus (RSV) seronegative cotton rats were immunized intramuscularly at four and eight weeks of age with either formalin-inactivated, alum-precipitated RSV grown in HEp-2 cell tissue cultures or virus-free HEp-2 cell tissue cultures similarly prepared. Sham-immunized animals served as controls. At 12 weeks of age, all animals were challenged with 6 x 10(5) plaque forming units of live RSV via the intranasal route. Animals were killed at predetermined days and evaluated for RSV antibody, virus replication and pulmonary histopathology. Of animals immunized with inactivated-RSV, 88% developed neutralizing antibody to RSV. Virus replication in the lungs of such animals was significantly reduced compared with tissue-culture-immunized animals. Surprisingly, however, both groups exhibited pulmonary histopathology, characterized by polymorphonuclear and mononuclear interstitial infiltrates. The virus-immunized animals manifested a more severe inflammatory reaction that reached a peak earlier than the virus-free, tissue-culture-immunized control group. In contrast, sham-immunized animals, when infected with live RSV for the first time, developed little or no pulmonary histopathology. The data suggest that the pathogenesis of pulmonary injury during natural RSV infection in the immunized host is due primarily to prior sensitization to the virus. In the animal model, sensitization to non-viral tissue culture components also contributes to lung injury.

Evaluation in non-human primates of the safety, immunogenicity and efficacy of recombinant vaccinia viruses expressing the F or G glycoprotein of respiratory syncytial virus.

It has been shown previously that immunization with recombinant vaccinia viruses expressing the F or G envelope glycoprotein of human respiratory syncytial virus (RSV) strain A2 induced a protective immune response in the lower respiratory tract of cotton rats against live RSV challenge. As a continuation of these studies, the safety, immunogenicity and efficacy of these recombinant vaccinia viruses was evaluated in non-human primates. Rhesus and patas monkeys were each inoculated intradermally at separate sites with the vaccinia-A2-F or vaccinia-A2-G recombinant or the parental vaccinia virus WR strain and the dermal lesion sizes were compared. Vaccinia-A2-F and vaccinia-A2-G recombinants produced lesions that were 5- to 15-fold smaller in area than vaccinia-WR. These studies indicated that insertion of either RSV gene into the thymidine kinase (TK) gene of vaccinia-WR significantly attenuated the virus for rhesus and patas monkeys. The immunogenicity of vaccinia-A2-F and vaccinia-A2-G was evaluated in squirrel, rhesus, African green, owl and patas monkeys. In four of the five species tested, the vaccinia-RSV recombinants stimulated levels of RSV serum-neutralizing antibodies considered to be protective for the lower respiratory tract of human infants and cotton rats. Interestingly, the level of RSV serum-neutralizing antibodies correlated with the size of the lesion. A boost in RSV serum-neutralizing antibody titres was not observed following a second inoculation. Owl monkeys inoculated with a single intradermal dose of vaccinia-A2-F and vaccinia-A2-G were completely resistant to infection of the lower respiratory tract with live RSV.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity and pathogenicity of temperature-sensitive modified respiratory syncytial virus in adult volunteers.

Single temperature-sensitive (ts) mutants of a subgroup A strain of respiratory syncytial (RS) virus whose multiplication is restricted at 39 degrees C in MRC-6 cells and double ts mutants that are restricted at 38 degrees C, were obtained following mutagenesis using 5-fluorouracil and acridine-like compounds. Isolation and propagation of the parental RSS-2 strain of RS virus and its derived ts mutants were carried out entirely in MRC-5 human diploid cells. The immunogenicity and disease-producing ability of four of these mutants and the parental unmodified strain have been assessed by intranasal administration into groups of about 20 adult volunteers. The results of these trials indicate that the capacity of the parental RSS-2 strain to produce upper respiratory tract infection in adults was not diminished by limited propagation in MRC-5 cells. The mutants on the other hand were impaired in this respect to varying extents. The double mutant tslB in particular has characteristics that suggest that it may be suitable for further development as a live RS virus vaccine. It retained near normal immunogenicity and replicative ability in the upper respiratory tract, while exhibiting greatly reduced disease-producing potential.

Response of vaccinated and unvaccinated bighorn sheep (Ovis canadensis canadensis) to experimental respiratory syncytial virus challenge.

Five Rocky Mountain bighorn sheep (Ovis canadensis canadensis), approximately 5 mo old and without detectable antibody titers to respiratory syncytial virus (RSV), were assigned to two groups to study the effects of RSV challenge inoculation in vaccinated (n = 3) and unvaccinated (n = 2) bighorns. The three lambs vaccinated with a modified live bovine RSV vaccine developed a detectable antibody response to the vaccine. Vaccinated and unvaccinated lambs challenged with an ovine isolate of RSV developed increased levels of neutralizing antibody, but clinical signs of disease were not observed. Neutralizing antibody titers to RSV remained higher (2-4-fold) in vaccinated lambs over time when compared to unvaccinated lambs.

Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice.

The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described. Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter. In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region. In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus. Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein. Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis. Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus. The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease.

Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.

Enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactiva ted virus

Cotton rats previously inoculated with Formalin-inactivated respiratory syncytial virus (RSV) were challenged intranasally with live RSV to induce an enhancement of RSV disease similar to that observed after the administration of Formalin-inactivated RSV vaccine to human infants 20 years ago. Within 24 h after infection with RSV, cotton rats developed pulmonary lesions that reached a maximum by day 4. Histologically, the lesions resembled an experimental pulmonary Arthus reaction. An action of Formalin on RSV appears to be responsible for this effect, because live virus or virus heated in the absence of Formalin did not induce enhanced immunopathology. Selected epitopes on the fusion (F) or attachment (G) or both RSV surface glycoproteins that are involved in inducing neutralizing antibodies were modified to reduce or ablate their antigenicity. However, other epitopes on the F or G or both glycoproteins were not ablated by Formalin, because cotton rats inoculated parenterally with a Formalin-inactivated virus developed a high level of F and G antibodies measurable by an enzyme-linked immunosorbent assay. At this time, the effect of Formalin on RSV cannot be localized to either the F or G glycoprotein of RSV.

Respiratory syncytial virus infection in immunosuppressed animals: implications in human infection.

Neonatal cotton rats were treated with cyclophosphamide parenterally for three weeks before intranasal inoculation of live respiratory syncytial virus (RSV). Immunosuppressive therapy resulted in severe depletion of lymphocytes from the peripheral circulation, the spleen, and the thymus. In contrast to normal rats, immunosuppressed animals developed severe pulmonary pathology with marked infiltration of foamy macrophages. Persistent degeneration and regeneration of bronchial epithelial cells were also observed, in which RSV antigens could be demonstrated by the immunoperoxidase technique. In addition, large quantities of live virus were recovered from the respiratory tract of these animals for as long as six weeks after infection. Systematic dissemination of RSV, which has never been documented in immunocompetent control rats, was found in four of the cyclophosphamide-treated animals. These results support clinical observations that cellular immunity may be very important in the pathogenesis of RSV infection in the human host.

Parenteral immunization with live respiratory syncytial virus is blocked in seropositive cotton rats.

Seropositive cotton rats cannot be immunized by intramuscular inoculation of live respiratory syncytial virus. Passively administered antiserum was shown to effect the immunosuppression. By contrast, the presence of serum antibody did not block immunization by the intranasal route.

Administration of a highly attenuated, live respiratory syncytial virus vaccine to adults and children.

A highly attenuated respiratory syncytial virus (RSV) experimental vaccine, RSV ts-2, was sequentially evaluated in adults, seropositive children, and finally, fully susceptible seronegative children. The vaccine was administered intranasally in doses ranging from 10(5.2) to 10(6.3) PFU/ml. In both adults and children, the vaccine proved to be poorly infectious. Although poor infectivity would not have been predicted from tissue culture studies of RSV ts-2 growth, the human experience closely parallels the experience in a series of animal models, including the chimpanzee. The poor infectivity of this RSV vaccine virus preparation suggests that the postulated defect in the RSV ts-2 fusion protein may be important in determining in vivo infectivity of RSV.

Secretory antibody response to respiratory syncytial virus infection.

The secretory antibody response to primary and secondary infection with respiratory syncytial virus (RSV) in Igg, IgM and 11S IgA immunoglobulin classes was determined using an indirect immunofluorescence antibody technique. Nasopharyngeal secretions were collected after naturally acquired primary or secondary infection with RSV. Immunoglobulin A responses to RSV were significantly greater in infants older than 6 months of age at the time of infection than in younger infants. Reinfection with RSV resulted in enhanced antibody production in all three immunoglobulin classes, and virus-specific antibody persisted for longer periods after secondary infection. Repeated immunizations with live RSV vaccine may be necessary for the development of immunity to severe illness.

Intramuscular inoculation of live respiratory syncytial virus induces immunity in cotton rats.

Intramuscular inoculation of infant or weanling cotton rats with 10(2.2) to 10(4) plaque-forming units of respiratory syncytial virus induced significant or complete resistance to infection in both the upper and lower portions of the respiratory tract. This resistance did not appear to be the result of in vitro neutralization of virus during homogenization of tissue. Virus was not recovered from the local site of inoculation after 5 min and was never detected in the respiratory tract of intramuscularly inoculated rats. Attempts to detect viral antigens at the site of inoculation by using indirect immunofluorescence were unsuccessful. However, inactivation of infectivity of three different strains of respiratory syncytial virus markedly reduced or completely ablated antigenicity and protective efficacy by the intramuscular route. This suggests that these viruses underwent limited replication, perhaps restricted to an abortive cycle, at the local site of inoculation. An immunosuppressive effect of passive maternally derived immunity was observed. Only 50% of weanling rats possessing passive maternal serum antibody were successfully immunized by intramuscular vaccination with live virus.

Further Investigations of Live Respiratory Syncytial Virus Vaccine Administered Parenterally

SummaryFurther clinical tests were carried out of a live attenuated respiratory syncytial virus vaccine administered parenterally. Neutralization of respiratory syncytial virus by homologous human antibody was found to be a complement-dependent event and addition of guinea pig complement greatly increased antibody titers and the sensitivity of the test. Ninety-seven percent of 116 initially seronegative children developed antibody following vaccination and there was no evidence for contagious spread of the infection. Clinical reactions, if any, were mild and inconsequential. A small portion of initially seropositive persons appeared to develop antibody by booster effect and a portion of initially seronegative and seropositive persons developed detectable nasal antibody. Long-term follow-up failed to reveal any increase in severe respiratory disease in initially seronegative persons who were vaccinated. A very substantial reservoir of seronegative persons was found in the 7 to 47 month age group (35%) reve...