Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."

Generation of atypical pulmonary inflammatory responses in BALB/c mice after immunization with the native attachment (G) glycoprotein of respiratory syncytial virus.

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.

Formulation of the purified fusion protein of respiratory syncytial virus with the saponin QS-21 induces protective immune responses in Balb/c mice that are similar to those generated by experimental infection.

The feasibility of employing a vaccine composed of the purified fraction 21 of Quillaja saponaria (QS-21) and the fusion (F) protein of respiratory syncytial virus (RSV) to induce protective immune responses in the lower respiratory tract of Balb/c mice was examined. Our goal was to compare local and systemic immune responses with those induced following immunization with the protein adsorbed to aluminium hydroxide (F/ALOH) adjuvant or by experimental infection. Sera from mice vaccinated with the QS-21 formulation (F/QS-21) contained elevated anti-F protein IgG antibody titres that were dependent on the dose of QS-21 employed. Similar to the immune responses generated by experimental infection, the sera from mice vaccinated with F/QS-21 possessed greater capacity to neutralize virus infectivity that was associated with the generation of heightened complement-fixing IgG2a antibody titres. In contrast, vaccination with F/ALOH elicited systemic immune responses that were characterized by a predominance of protein-specific antibodies of the IgG1 subclass and lower neutralizing antibody titres. The capacity of F/QS-21 to facilitate local pulmonary immune responses was also examined and found to be similar to those induced by experimental infection. After virus challenge, a 90-fold increase in the number of F protein-specific antibody-secreting cells was observed and associated with the clearance of virus from the infected lungs. Moreover, elevated levels of antigen-dependent killer cell activity were detected and appeared to be mediated by class I major histocompatibility complex restricted CD8+ T cells. Additional characterization of the pulmonary immune response was performed on the cellular infiltrates obtained after bronchoalveolar lavage and on formalin-fixed lung tissue. The local protective immune responses induced after challenge of the groups immunized with F/QS-21 or infectious virus were significantly different from those elicited in naive control mice injected with adjuvant alone, or in mice immunized with F/ALOH. The cellularity of the lavage fluids from the former groups was characterized by a significantly greater percentage of lymphocytes and less neutrophils. In similar fashion histological evaluation of the lungs from mice immunized with F/QS-21 or infectious virus revealed significantly elevated local immune responses after challenge. In conclusion, the results suggest that formulation with F/QS-21 alters the qualitative and quantitative nature of the immune response to the F glycoprotein when compared with the traditional aluminium-based adjuvants.

Safety and immunogenicity of a subunit respiratory syncytial virus vaccine in children 24 to 48 months old.

A subunit vaccine for respiratory syncytial virus (RSV) consisting of purified fusion glycoprotein (designated PFP-1) was tested in children 24 to 48 months old. Two doses of 20 micrograms (n = 13) and 50 micrograms (n = 10) were compared with a saline (n = 24) placebo control group. Local and systemic reactions, reported within 96 hours postvaccination, were mild, transient, and did not differ significantly from the control cohort. Long term follow-up through at least one, and in some cases two, RSV seasons showed no serious RSV illness in vaccinees at any time. There was, therefore, no evidence of disease enhancement postvaccination. In the 20-micrograms cohort, 92% responded to vaccination by a 4-fold increase in enzyme-linked immunosorbent titer to the F glycoprotein and 42% had a 4-fold or greater rise in neutralizing titer to the A2 virus. In the 50-micrograms cohort 100% responded by enzyme-linked immunosorbent to the F glycoprotein and 70% responded by A2-neutralizing titers. The neutralizing titers in the vaccinated cohorts were similar to those seen previously in adults. These data show the ability of the subunit vaccine to boost existing immunity and to prime for a response to natural virus exposure in children who were seronegative at the time of vaccination.

Attenuated temperature-sensitive respiratory syncytial virus mutants generated by cold adaptation.

Two strains of respiratory syncytial virus (RSV), RSV 2B and RSV 3A (representing subgroup B and A virus respectively) were cold-adapted by passaging in Vero cells for up to 42 weeks at successively lower temperatures down to 20 degrees C. Successful cold adaptation of the virus population was dependent on the amount of time the cultures were maintained at the various low temperatures, as well as on the strain of virus used. Temperature-sensitive (TS) mutants appeared in the cold passaged virus populations; however, the majority of the virus variants remained predominantly non-TS. Four RSV 2B and three RSV 3A TS mutants were selected for further characterization. These seven TS mutants retained their fusion phenotype and two major neutralizing antibody epitopes, and displayed varying levels of temperature sensitivity. Six of the seven mutants had a cold-adapted (CA) phenotype. All of the RSV 2B mutants were highly attenuated in cotton rats and two of the mutants elicited relatively high levels of neutralizing antibody and were able to protect rats against virus challenge. The RSV 3A TS mutants grew well in the nose but poorly in the cotton rat lungs, as did the parental 3A virus. All 3A mutants elicited high titers of neutralizing antibody and provided complete protection against virus challenge. These mutants showed varying levels of temperature sensitivity in vitro and attenuation in vivo and represent potential vaccine candidates.

The pulmonary immune response of Balb/c mice vaccinated with the fusion protein of respiratory syncytial virus.

We have investigated the efficacy of vaccination with the purified fusion (F) protein of respiratory syncytial virus (RSV) on aluminium hydroxide adjuvant in Balb/c mice. The purpose of the study was to define the role of the local pulmonary mononuclear cell (PMC) infiltrate in the clearance of virus from the lower respiratory tract. Balb/c mice immunized with F protein were able to inhibit the replication of virus in the lungs as early as 4 days after intranasal challenge. In contrast, unimmunized mice required 8 days. Examination of humoral immune mechanisms demonstrated that vaccination with the purified protein induced moderate titres of serum neutralizing antibody. In addition, immunization induced low to moderate levels of antigen-dependent killer cell activity. To examine the immunological events responsible for virus clearance in vivo, PMC infiltrates were isolated after virus challenge and tested directly for protective capacity. After virus challenge, the F protein-immune mice were able to recall the cytolytic cells to the pulmonary tissues. The results further suggested that the local antigen-dependent killer activity was mediated by cytolytic T cells of the CD8 phenotype. Adoptive transfer studies were also conducted to identify further the role the PMC infiltrate had in protective immunity. Adoptive transfer of F protein-educated PMC into naive syngeneic recipients suggested that the pulmonary infiltrates contained the cellular constituents necessary for protective immunity. Both humoral and cellular immune elements were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced pulmonary pathology associated with the use of formalin-inactivated respiratory syncytial virus vaccine in cotton rats is not a unique viral phenomenon.

The specificity of viral antigens in the formalin-inactivated, alum-precipitated respiratory syncytial virus (FI-RSV) vaccine in augmenting the pulmonary inflammatory response was evaluated. Cotton rats were immunized with a FI-RSV vaccine derived from Vero cells, a monkey cell line, or HEp-2 cells, a human cell line. The FI-RSV/Vero and the FI-RSV/HEp-2 vaccines were prepared similarly to the original Lot-100 FI-RSV vaccine that was associated with enhanced disease in the mid-1960s field trials. Each vaccine was administered intramuscularly at various doses and intervals. At 1, 4 or 7 weeks after the last vaccine dose, cotton rats were challenged with 10(6) plaque-forming units of live RSV grown in HEp-2 cells. For controls, FI-parainfluenza, FI-HEp-2 and alum vaccines, and live RSV primary infection were used. For measuring virus replication and histopathology, lungs were harvested at 4 and 8 days postchallenge. A dose-response relationship to vaccine dose was observed for ELISA, neutralizing and antifusion antibodies. All animals given three doses or two of the higher doses of FI-RSV/Vero vaccine developed significant neutralizing antibody, were protected against pulmonary virus replication and had similar low levels of histopathology compared with live RSV and controls. Two immunizations of the lowest dose of FI-RSV/Vero vaccine did not induce neutralizing antibody, did not provide protection of the lung against RSV and did not enhance the pulmonary cellular response. However, FI-RSV/HEp-2 vaccine was associated with significant enhanced pulmonary histopathology despite inducing high titres of neutralizing antibody and protecting the lungs against RSV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of parental relaxation training on glycosylated hemoglobin of children with diabetes.

Since 1975, interest in biofeedback, relaxation and self-regulation has increased for people with diabetes mellitus. Although children and adults using relaxation training enhanced by some method of biofeedback training have been studied, nothing has been done to document its effect on the children's Hgb A1c (glycosylated hemoglobin). Parents scoring 300 or above on the "Life Stress Scale" participated in 10 weeks of progressive relaxation training supported by temperature biofeedback. Their children's blood was tested for glycosylated hemoglobin prior to the relaxation training process and again at the conclusion of the study. The outcome demonstrated no significant differences in pre- versus post-Hgb A1c in the control group (P less than 0.16). In spite of the small sample size, the pre- versus post-experimental group demonstrated a significant difference (P less than 0.045). The significance of the utilization of such practices will be discussed relative to the outcome of increased control of blood glucose levels.

Changes in cortical functioning with acute hypoglycemia and hyperglycemia in type I diabetes.

In this study, 18 type I (insulin-dependent) diabetic subjects aged 22-35 yr (mean age 29.3) and within 10 yr of diagnosis (mean 7.7) performed a battery of cognitive and psychomotor tasks under conditions of hypoglycemia (50 mg/dl), normoglycemia (100 mg/dl), and hyperglycemia (300 mg/dl). Blood glucose levels during testing were precisely maintained at the preselected level via a Biostator insulin/glucose-infusion system. The order of glycemic level was counterbalanced across subjects in a single-blinded design. Performance on tasks requiring visual tracking, visuomotor speed, concentration, and planning ability (pursuit rotor and trails B) were significantly impaired under conditions of hypoglycemia compared with normoglycemic levels. Visual reaction time was not significantly impaired under conditions of hypoglycemia or hyperglycemia.

Cerebral palsy and stereotactic neurosurgery: long term results.

A retrospective study was performed on a group of 28 patients with cerebral palsy, who had undergone a stereotactic encephalotomy for hyperkinesia or dystonia. The mean postoperative follow up period was 21 years (range: 12-27). Eighteen patients were available for follow up, nine had died, and one could not be traced. A positive result was obtained in eight of the 18 reassessed patients. Determining factors for the outcome were the degree of preoperative disability, side effects of the operation, and ageing since operation. The more favourable results were obtained in patients with hyperkinesia, tremor, and predominantly unilateral dystonia.

Inhibition of cytomegalovirus replication by smooth-muscle relaxing agents.

The effect of the smooth-muscle relaxing agents, papaverine, sodium nitroprusside, and verapamil, on the replication of human cytomegalovirus (CMV) was investigated. At a concentration of 100 microM, infectious yields of CMV were reduced by 1.23 to 5.72 log10 by these drugs (papaverine, 5.72 log10; nitroprusside, 1.85 log10; verapamil, 1.23 log10). The ED50 for papaverine was found to be somewhat less than 1 microM, a concentration which appears to be within the range achieved clinically. Papaverine did not irreversibly modify treated cells to a virus-resistant state since treatment of cells with papaverine from 24 hr before until immediately prior to CMV infection did not significantly reduce CMV yields. Replication of CMV was most sensitive to inhibition when papaverine was added at or before 6 hr after CMV infection. Addition of papaverine at later times resulted in a substantial reduction of the inhibitory effect on virus yields, suggesting that the phase of CMV replication sensitive to papaverine inhibition occurred early in the replication cycle. These results, particularly in light of the potency of papaverine, indicate that some smooth-muscle relaxing agents have significant antiviral activity toward the replication of CMV.

Kinetics of the interaction of monophosphates of the antiviral nucleosides 2'-fluoro-1-beta-D-arabinofuranosylpyrimidine and (E)-5-(2-bromovinyl)-2'-deoxyuridine with thymidylate kinases from Vero cells and herpes simplex virus types 1 and 2.

The affinities of the monophosphates of 2'-fluoro-5-iodo-1-beta-D-arabinofuranosyluracil and its 5-methyl analog for cellular thymidylate kinase were two or more orders of magnitude greater than for the thymidine-thymidylate kinases from herpes simplex virus types 1 and 2. In contrast, the monophosphate of (E)-5-(2-bromovinyl)-2'-deoxyuridine was found to have a higher affinity for the viral enzymes than for the cellular enzyme.

Reduction of 4-nitroquinoline 1-oxide to 4-hydroxyaminoquinoline 1-oxide in lysates of cytomegalovirus-infected cells.

The rates of virus inactivation by 4-nitroquinoline 1-oxide (NQO) and 4-hydroxyaminoquinoline 1-oxide (HAQO) were compared and samples of cytomegalovirus (CMV)-infected cell lysates to which NQO had been added were examined for the presence of HAQO. These experiments demonstrated that (i) CMV inactivation by HAQO was more rapid than with NQO, (ii) virus inactivation by either NQO or HAQO failed to demonstrate a photodynamic component, and (iii) NQO-treated stocks contained HAQO, indicating reduction of NQO to HAQO. The results support the concept that metabolism of NQO to HAQO enhances the genotoxic effect of NQO.

Similarities between cytomegalovirus-induced cell rounding and contraction of smooth muscle cells.

Cytomegalovirus infection of human fibroblastic cell cultures resulted in rounding of the cells and a subsequent decrease in their size. Smooth muscle relaxing agents such as papaverine, hydralazine, diazoxide, nitroprusside and verapamil blocked these early cytopathogenic effects suggesting that cell rounding results from a contractile-like response involving an influx of calcium ions.

Herpes virus inactivation by chemical carcinogens: differential inactivation of herpes simplex viruses by 4-nitroquinoline 1-oxide and related compounds.

Treatment of stocks of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with the chemical carcinogen 4-nitroquinoline 1-oxide (NQO) resulted in inactivation of virus infectivity at rates which were directly dependent on the concentration of NQO and interval of exposure to NQO. HSV-1 strains were more sensitive than HSV-2 strains to inactivation by NQO, although survival curves of both HSV types were multicomponent. Exposure of HSV-2 to a related group of chemicals suggested that the structural specificity required for inactivation of this virus was similar to that established by previous in vivo carcinogenicity tests.

Correlation of the chemical structure of 4-nitroquinolines inactivating human cytomegalovirus and established in vivo carcinogenicity tests.

Inactivation of the infectivity of human cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) has been observed following exposure to 4-nitroquinoline 1-oxide (NQO) or its metabolite, 4-hydroxyaminoquinoline 1-oxide (HAQO). The present study of the specificity of the chemical structure of 4-nitroquinolines demonstrated that both the 4-nitro and 1-oxide groups were required for inactivation of virus infectivity. Reduction of the 4-nitro group to a 4-hydroxyamino group enhanced activity, while further reduction to an amino group resulted in loss of activity against virus infectivity. The capacity to inactivate virus was also lost by substitution of the pyridine ring for the quinoline nucleus of NQO. The relationship between the chemical structure and the ability to inactivate viruses studied here correlates well with earlier in vivo carcinogenicity studies of the same group of chemicals.

Inactivation of human cytomegalovirus by the chemical carcinogen 4-nitroquinoline 1-oxide.

The infectivity of cytomegalovirus (CMV), strain Davis, was inactivated by 4-nitroquinoline 1-oxide (NQO). A series of survival curves indicates that the rate of inactivation was directly dependent on the concentration of NQO over a range of 5 to 200 microgram/ml. At concentrations of I microgram/ml or less, inactivation of virus stock was not observed and at concentrations in excess of 200 microgram/ml, the cellular toxicity of residual NQO prevented quantification of the relatively low surviving infectivity. At a concentration of 200 microgram/ml NQO or less, the loss of virus infectivity could be clearly shown to result from the interaction of NQO with virus and not with cells, since the addition of similar doses of NQO to assay cells simultaneously with virus did not adversely affect the sensitivity of the assay cells to measure virus infectivity. Similarly, the dimethylsulphoxide carrier at concentrations of 5% or less was shown to have a negligible effect on both virus infectivity and on the sensitivity of human skin muscle cells to assay virus infectivity. NQO inactivation of virus infectivity appeared to depend very little on white light, since the kinetics of inactivation in the presence and in the absence of white light were similar.