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Biomedical subjects

E J Stott

Publications and source records attributed to E J Stott.

At least 55 records · Page 3Linked to original sources

Field trial of a quadrivalent vaccine against calf respiratory disease.

A quadrivalent vaccine containing the killed antigens of respiratory syncytial virus, parainfluenza virus type 3, Mycoplasma dispar and M bovis, emulsified with an oil adjuvant, was tested for efficacy against naturally occurring calf respiratory disease. Three batches of beef cattle aged 12, seven and three weeks at the time of first vaccination were used. Within each batch of approximately 100 animals, half were vaccinated subcutaneously on three occasions, three weeks apart and half served as unvaccinated controls. Over the trial period, from November 1981 to May 1982, 27 per cent of the control calves were treated for respiratory disease compared with 16.3 per cent of the vaccinated animals. This reduction of non-fatal disease in the vaccinated animals represented a protection rate of almost 40 per cent and was statistically significant (P less than 0.05). Mortality was also reduced from 3.4 per cent in the control calves to 1.9 per cent in the vaccinated animals but this difference was not statistically significant. During a major outbreak of disease associated with respiratory syncytial virus, the protection rate increased to 69 per cent (P less than 0.01). Furthermore, in the batch of cattle aged seven weeks at first vaccination there was significantly less pneumonic consolidation at death in the vaccinated animals than in the control animals (P less than 0.05).

Animals↗

Lymphocyte transformation response of calves to respiratory syncytial virus.

Virus-specific cell-mediated immunity, as determined by in vitro lymphocyte transformation (LT), was demonstrated in calves following infection and vaccination with respiratory syncytial virus (RSV). After experimental infection, 4 of 6 gnotobiotic calves and 6 of 21 conventional calves developed a significant LT response to RSV. By means of a whole blood assay, the LT responses of calves were examined after vaccination with an inactivated vaccine, which consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with a bovine strain of RSV (GC), a live modified bovine strain of RSV (MV), or a live temperature-sensitive mutant of a human strain of RSV (ts-l). Three weeks after vaccination, a virus-specific LT response was detected in 6 of 6 calves given the GC vaccine, 0 of 4 calves given the MV vaccine, and 2 of 4 calves given the ts-l vaccine. The magnitude of the response was greatest in those animals given the GC vaccine. There was no significant correlation between the magnitude of the LT response and levels of serum neutralising antibody. However, the LT response did correlate with serum antibody measured by the single radial haemolysis test 3 weeks after the first vaccination. LT activity to RSV was associated with T and not B lymphocytes. The development of a virus-specific LT response in calves given an inactivated RSV vaccine was not associated with an increase in respiratory disease following challenge with live virus, but rather was related to increased resistance to RSV infection.

Animals↗

Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.

Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgG1, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity. Finally, this report describes histological examination of mouse lungs after vaccination and challenge. Vaccinated mice that were subsequently challenged had significantly greater lung lesion scores than unvaccinated challenged mice. The lesions were primarily peribronchiolar and perivascular infiltrations of polymorphonuclear cells and lymphocytes. Further work will establish whether these pulmonary changes are a desirable immune response to virus invasion or a potential immunopathogenic hazard. The results have important implications for planning a strategy of vaccination against RS virus and emphasize potential dangers that may attend the use of recombinant VV as vaccines.

Administration, Cutaneous↗

Expression of the fusion protein of human respiratory syncytial virus from recombinant vaccinia virus vectors and protection of vaccinated mice.

Vaccinia virus (VV) recombinants were constructed that contained full-length cDNA copies of the fusion (F) protein gene of human respiratory syncytial (RS) virus. The F protein gene was placed next to the strong early-late VV 7.5-kilodalton promoter and was located within the VV thymidine kinase (tk) gene. Full-length recombinant transcripts that initiated at both the tk and the 7.5-kilodalton promoters accumulated in cells early in infection, and one or more of these transcripts was translated to yield a glycoprotein which comigrated with Fo, the fusion protein precursor. This precursor was processed by proteolytic cleavage to produce the two disulfide-linked subunits F1 and F2, which were both glycosylated and of the same electrophoretic mobility as authentic F1 and F2. Immunofluorescence studies demonstrated that the mature F protein was transported to and expressed on the surface of recombinant VV-infected cells. Inoculation of rabbits with a recombinant vector expressing F resulted in the production of antiserum specific for the RS virus F protein. This antiserum neutralized virus infectivity and was capable of preventing fusion in RS virus-infected cells. Mice were vaccinated with recombinants expressing the F protein. At 3 weeks postinoculation, these animals had serum antibody against RS virus F protein. At 5 days after intranasal challenge with RS virus, the lungs of the mice previously vaccinated with recombinants expressing F protein were free of detectable RS virus, whereas the lungs of unvaccinated mice contained 10(4.2) PFU of virus per g.

Cell Line↗

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.

Animals↗

Clearance of persistent respiratory syncytial virus infections in immunodeficient mice following transfer of primed T cells.

Little is known of the role of T-cell mediated immune responses in the clearance and pathogenesis of respiratory syncytial virus (RSV) infection. In this study, we established persistent pulmonary RSV infections in athymic nu/nu BALB/c mice or immunodeficient irradiated BALB/c mice, and examined the patterns of virus clearance following adoptive transfer of splenic memory T cells. Primed T cells transferred between Day 5 and Day 8 of infection will clear lung RSV from both nu/nu mice and irradiated mice within 10 days of transfer. Partially purified Lyt 2+ T cells are more effective than L3T4+-selected T cells. No RSV-specific serum antibody could be detected, suggesting that clearance is by an antibody-independent mechanism. In contrast, delayed (Day 14) transfer of primed L3T4+-selected cells clears lung RSV from nu/nu mice, and this correlates with RSV-specific serum antibody production. Clearance is not seen following Day 14 transfer of total primed T cells or T cells selected for the Lyt 2+ subset.

Animals↗

The detection of respiratory syncytial virus in nasopharyngeal aspirates: assessment, formulation, and evaluation of monoclonal antibodies as a diagnostic reagent.

Comparisons were made between standard methods of cell culture, indirect immunofluorescence (IF) using hyperimmune respiratory syncytial virus (RSV) antiserum, and indirect IF using mouse monoclonal antibodies directed against various epitopes of RSV for the detection of RSV in nasopharyngeal aspirates. The monoclonal antibodies were used singly and in pools of different specificities which in turn were tested in both direct and indirect IF. In a preliminary study, aspirates from 227 infants were examined for RSV by standard methods. The results were compared with the detection of RSV in these aspirates using nine separate monoclonal antibodies and a pool consisting of five monoclonal antibodies. Respiratory syncytial virus was detected in 64 (28%) by cell culture, in 68 (30%) by indirect IF using bovine polyclonal antibody (BPA), and in 75 (33%) by indirect IF using the monoclonal antibody pool. The nine individual monoclonal antibodies when tested separately were less sensitive, detecting between 8 and 77% of all aspirates found to be positive by culture. After statistical analysis of the results obtained in the preliminary study, a refined monoclonal antibody pool was prepared and in a further study was tested by both direct and indirect IF in parallel with our two standard methods. Slides prepared from 303 nasopharyngeal aspirates collected between 1981 and 1984 and either tested the same day or stored at -20 degrees C were used to evaluate these reagents. Overall agreement between the four tests was found in 274 (90%) specimens. Cell culture detected RSV in 68 (22%) specimens, indirect IF with BPA in 67 (22%), indirect IF with monoclonal antibody in 72 (24%), and direct IF with monoclonal antibody in 79 (26%). The pool of monoclonal antibodies used in direct or indirect IF was thus more sensitive than our standard methods for the detection of RSV in nasopharyngeal aspirates, and direct IF tests could be completed in 40 minutes.

Adult↗

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.

Animals↗

Mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus.

Mycoplasma bovis was inoculated alone or in combination with respiratory syncytial virus into the respiratory tracts of 12 gnotobiotic calves. Clinical signs ranged from transient pyrexia to protracted fever accompanied by severe lower respiratory signs and in one case, arthritis. Pulmonary lesions included foci of coagulative necrosis surrounded by mononuclear cells and suppurative bronchiolitis with varying degrees of lympho-reticular hyperplasia. No enhancement of lesions occurred in the combined infections of M. bovis and respiratory syncytial virus. M. bovis was identified by immunoperoxidase labelling in lesions of necrosis, especially at interfaces between the lesion and mononuclear cells and in bronchiolar exudates. Organisms were also located in necrotic lesions of joint capsules, in tonsillar crypts, and in liver.

Animals↗

Cytotoxic lymphocytes in the lungs of mice infected with respiratory syncytial virus.

Mice infected with respiratory syncytial virus (RSV) developed cytotoxic lymphocytes in the lungs, which lysed RSV-infected, but not uninfected cells. Cytotoxic activity was greatest 7 to 9 days after infection, was virus-specific, MHC-restricted and abolished by treatment of lymphocytes with anti-Thy 1.2 or with anti-Lyt 2.2 sera and complement. There was a close temporal relationship between the appearance of these cytotoxic lymphocytes in the lung and clearance of virus. In contrast, RSV persisted in the lungs of athymic (nude) mice and such animals failed to develop RSV-specific cytotoxic lymphocytes. Thus, cytotoxic T-cells may have an important role in recovery from RSV infection.

Animals↗

Infection of gnotobiotic calves with a bovine and human isolate of respiratory syncytial virus. Modification of the response by dexamethasone.

A bovine and a human strain of RSV both adapted to bovine cell culture, have been inoculated separately into 13 and 7 gnotobiotic calves respectively by 3 different methods. Both strains infected calves and showed similar growth patterns. Virus was recovered from the nasopharynx between one and 11 days with peak titres between 3 and 8 days following inoculation. With the exception of 4 calves treated with dexamethasone no clinical signs and only minimal macroscopic lesions of the lung were induced, which histologically comprised a mononuclear infiltration of alveolar walls and of the peribronchiolar tissue. The serological response to both strains was similar. Antibody was detected by virus neutralisation or single radial haemolysis from 12 days after inoculation. Specific anti-RSV IgM was detected from 10 days and IgG from 16 days after inoculation. Treatment with dexamethasone (0.5 mgm/Kg daily for 10 days) enhanced lung lesions produced by the bovine strain, prolonged the period of virus shedding and increased peak titres. The specific IgM response was suppressed.

Animals↗

A comparison of three vaccines against respiratory syncytial virus in calves.

An inactivated vaccine against respiratory syncytial virus (RSV) was compared with two live vaccines. The inactivated (GC) vaccine consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with RSV and emulsified with oil adjuvant. The live vaccines were a modified virus (MV) derived from a bovine strain of RSV and a temperature-sensitive mutant (ts-1) derived from a human strain. The GC vaccine was inoculated subcutaneously into 12 calves and the live vaccines intramuscularly into eight calves each. Nine unvaccinated calves acted as controls. The vaccines were administered in two doses 3 weeks apart and all calves were challenged intranasally with 2 X 10(7) p.f.u. of bovine RSV 3 weeks after the second dose. At the time of challenge calves given GC, MV and ts-1 vaccines had mean serum neutralizing antibody titres of 25, 19 and 2 respectively; mean titres of IgG1 antibody by radioimmunoassay were log10 4.5, 1.3 and 2.6 respectively and mean zone areas by single radial haemolysis (SRH) were 107, 27 and 36 mm2 respectively. Eleven of 12 calves given GC vaccine were completely protected against challenge but all control animals and those given the two live vaccines were infected. The mean peak titre of virus in nasal swabs of control calves was 3.0 log10 p.f.u./ml and the mean duration of virus shedding was 6.8 days. Both these parameters were significantly reduced in animals given MV and ts-1 vaccines: mean peak titres were 2.1 and 2.4 log10 p.f.u./ml and mean duration of shedding was 3.4 and 3.3 days respectively. Thus, protection correlated better with RSV antibody detected by radioimmunoassay and SRH than with neutralizing antibody. These results are discussed in relation to the possible mechanism by which protection was mediated.

Animals↗

Respiratory syncytial virus infection in mice.

The A2 strain of human respiratory syncytial virus replicated in the nose and lung of BALB/c mice, with virus growing to higher titers in older animals than in younger animals. Virus was recovered from the nose between days 2 and 7 with peak titers on days 3 and 4, and from the lungs between days 2 and 9, with peak titers on days 4 through 6. Serum antibody developed 2 weeks after infection. Viral antigen was demonstrated in the alveolar cells of the lung by immunofluorescence. Histopathological changes included infiltration by mononuclear cells of the peribronchiolar and perivascular tissue, some interstitial thickening, and formation of multinucleated giant cells. Virus could not be recovered from the respiratory tract of mice inoculated with bovine strains of respiratory syncytial virus. Growth of the A2 strain of human respiratory syncytial virus in different cell lines affected its infectivity for mice. Infection of BALB/c mice with respiratory syncytial virus provides a highly reproducible model for the study of the pathogenesis of and mechanisms of immunity to this virus.

Age Factors↗

The characterization and uses of monoclonal antibodies to respiratory syncytial virus.

Fifteen hybridomas secreting monoclonal antibodies against respiratory syncytial virus (RSV) have been produced. Three react with nucleoprotein (MW 42 000 daltons), 1 with phosphoprotein (MW 36 000 daltons), 1 with a larger protein (MW 60 000 daltons) and 10 with the fusion glycoprotein (MW 46 000 + 22 000 daltons). By immunofluorescent staining of infected cells, 5 monoclonal antibodies bind to cytoplasmic inclusions and the remainder give diffuse cytoplasmic fluorescence. Virus infectivity is neutralized by two monoclonal antibodies. Thirteen react with both bovine and human strains of RSV, two fail to react with three human strains. The antibodies have been used to induce passive protection in mice against challenge with RSV and to purify viral components on immunoadsorbent columns.

Animals↗

Monoclonal antibodies protect against respiratory syncytial virus infection in mice.

Twenty-five monoclonal antibodies (Mab) to respiratory syncytial virus (RSV) and two to hepatitis B virus were inoculated intravenously into mice. Twenty-four hours later the mice were challenged intranasally with RSV. Eleven of 14 Mab against fusion protein and four out of six Mab against a larger glycoprotein (GP84) significantly reduced the titre of RSV in the lungs when mice were killed 5 days later. Five Mab against three other RSV proteins and two Mab against hepatitis B virus had no significant effect on RSV infection. These results indicated that serum IgG against one epitope on the fusion protein and another on the larger glycoprotein (GP84) will completely protect mice against challenge. These epitopes are primary candidates for an RSV vaccine produced by techniques of gene cloning and peptide synthesis.

Animals↗