RESUMEN
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.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Humano/inmunología , Vacunación , Proteínas Virales de Fusión/inmunología , Preescolar , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Masculino , Pruebas de Neutralización , Virus Sincitial Respiratorio Humano/aislamiento & purificaciónRESUMEN
Enzyme immunoassays (EIAs) producing either chromogenic or fluorogenic end products were developed and evaluated for detection of eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses in pools of Aedes triseriatus mosquitoes. Overnight incubation of the mosquito samples in the EIA significantly enhanced the sensitivity of the test. Both the EEE and HJ EIAs were sensitive, readily detecting one infected mosquito in a pool with 99 noninfected, and specific, distinguishing homologous from the alternate alphavirus and other arboviruses. By 3 days post-infection after intrathoracic inoculation, EEE virus was isolated from 100% (30/30) of the mosquitoes examined. Concurrently, EEE virus antigen was detectable by EIA in 100% (30/30) of examined mosquitoes and by indirect fluorescent antibody (IFA) technique in 77% (23/30) of the examined mosquito head squash preparations.
Asunto(s)
Aedes/microbiología , Alphavirus/inmunología , Antígenos Virales/análisis , Virus de la Encefalitis Equina del Este/inmunología , Técnicas para Inmunoenzimas , Animales , Antígenos Virales/aislamiento & purificación , Virus de la Encefalitis Equina del Este/aislamiento & purificación , Ratones , ConejosRESUMEN
The timing and sequence of eastern equine encephalitis (EEE) virus replication was studied in the organs from a colony strain of orally infected Culiseta melanura. Three methods of virus assay were used: fluorescent antibody (FA) staining of dissected organs; virus titration in cell culture of whole mosquitoes, dissected organs, hemolymph, and egg rafts; and transmission electron microscopy (TEM) of infected hindguts. EEE virus replicated rapidly in Cs. melanura, first in the posterior midgut, after which it disseminated into the hemocoel where hemolymph transported virus to other organs causing a systemic infection that eventually involved all organs examined, except ovarioles. No initial decrease in virus titer of whole mosquitoes or dissected organs was observed when mosquitoes were collected at daily intervals. Muscle tissue contained the greatest amount of specific fluorescence and the largest aggregates of virus that were visible by TEM. Dissemination of virus occurred rapidly, in some mosquitoes after less than or equal to 17 hours of extrinsic incubation (EI). All infected mosquitoes had disseminated infections after 3 days of EI. Maximum amounts of virus were obtained from whole mosquitoes on the 7th day of EI. FA staining of hindguts was determined to be a rapid and reliable method for detection of EEE virus dissemination and replication in Cs. melanura.
Asunto(s)
Alphavirus/crecimiento & desarrollo , Culicidae/microbiología , Virus de la Encefalitis Equina del Este/crecimiento & desarrollo , Insectos Vectores/microbiología , Animales , Antígenos Virales/análisis , Sistema Digestivo/microbiología , Virus de la Encefalitis Equina del Este/inmunología , Virus de la Encefalitis Equina del Este/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente , Ganglios/microbiología , Hemolinfa/microbiología , Músculos/microbiología , Óvulo/microbiología , Glándulas Salivales/microbiología , Factores de TiempoRESUMEN
Enzyme immunoassays (EIAs) for eastern equine encephalomyelitis (EEE) and Highlands J (HJ) virus antigens were compared in a retrospective study with standard virus isolation procedures (VIP) for detection of alpha virus-infected mosquito pools. The original VIP was a plaque assay in chick embryo cell culture, and was performed in the years from 1979 to 1981. Using the original VIP as the reference standard, the sensitivity rate of the EIA was 0.7674 and the false negative rate was 0.2326. However, when the storage age and the initial virus titer of the sample were considered, the sensitivity rate increased. For samples containing greater than 1,500 plaque-forming units (PFU) per ml of virus during the original VIP, the sensitivity rate of the EIA was 0.97; but the rate declined to 0.14 for those originally containing less than 500 PFU per ml. Most of the false negatives (68%) occurred with samples containing less than 500 PFU per ml. Presumably the low quantities of virus in these 50 pools were lost during storage and handling; virus was obtained from only 16% (8/50) during reisolation attempts using BHK-21 cells. Specificity of the EIA was excellent; no false positive results were obtained and serological identification was identical to that determined by plaque reduction neutralization in greater than 98% of the pools examined. Characteristics of the pools, such as pool size, species of mosquitoes, or gravidity did not affect the EIA results. These studies support the use of EIAs in surveillance programs attempting to determine infection rates of known arboviruses in vector populations.
Asunto(s)
Alphavirus/inmunología , Antígenos Virales/análisis , Culicidae/microbiología , Virus de la Encefalitis Equina del Este/inmunología , Técnicas para Inmunoenzimas , Alphavirus/aislamiento & purificación , Animales , Virus de la Encefalitis Equina del Este/aislamiento & purificaciónRESUMEN
The 14 & 6 Hz positive spike phenomenon is generally considered a normal variant finding. Our experience prompted this re-evaluation, which consisted of three parts: In children referred for sleep electroencephalograms (EEGs), 100 children with normal EEG and 100 with 14 & 6--the 14 & 6 correlated with behavior disorder and aggression; In 75 children referred for neurological evaluation and EEG because of behavior problems, 52% had 14 & 6 (excluding those with paroxysmal EEGs); and In 57 symptomatic children having prominent 14 & 6, tabulation of symptoms yielded a complex but coherent clinical picture, including disturbances of temper, mood, attention, learning, and sleep. We conclude that 14 & 6 has clinical associations and deserves study.
Asunto(s)
Electroencefalografía , Epilepsia/diagnóstico , Trastornos Neurocognitivos/diagnóstico , Fases del Sueño , Adolescente , Niño , Trastornos de la Conducta Infantil/diagnóstico , Trastornos de la Conducta Infantil/tratamiento farmacológico , Preescolar , Epilepsia/tratamiento farmacológico , Potenciales Evocados/efectos de los fármacos , Femenino , Humanos , Masculino , Trastornos Neurocognitivos/tratamiento farmacológico , Psicotrópicos/uso terapéutico , Fases del Sueño/efectos de los fármacosRESUMEN
La Crosse (LAC) viral antigen was detected in the skin of inoculated mice. Antigen was detected principally in the dermis of 102 of 120 (85%) mice with clinical signs of illness. To demonstrate the specificity of the fluorescence, LAC virus was isolated from selected samples and was identified by the complement-fixation test. Antigen was most often detected in skin rich in vascular and nerve tissue and was probably disseminated by hematogenous spread. Antigen was found in muscle, vascular, nervous, and other tissues of the dermis, depending on the age of the mice. Antigen was first detected in the skin of 80% of the mice (5 to 6 days of age) on postinoculation day (PID) 3. On PID 4, 100% of these mice were positive, but on PID 5, only 40% were positive, indicating that clearance or neutralization of antigen had occurred in peripheral areas. The skin biopsy technique may be applicable to diagnosis of arboviral infections in other vertebrates.
Asunto(s)
Antígenos Virales/análisis , Bunyaviridae/inmunología , Virus de la Encefalitis de California/inmunología , Encefalitis por Arbovirus/inmunología , Encefalitis de California/inmunología , Piel/inmunología , Animales , Encefalitis de California/diagnóstico , RatonesAsunto(s)
Antígenos Virales/inmunología , Proteína HN , Proteínas Virales de Fusión/inmunología , Proteínas Virales , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Humanos , Mapeo Peptídico , Conejos , Infecciones por Respirovirus/inmunología , Proteínas del Envoltorio ViralRESUMEN
An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19-20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.
Asunto(s)
Bordetella pertussis/inmunología , Técnicas de Laboratorio Clínico/normas , Tos Ferina/diagnóstico , Tos Ferina/prevención & control , Centers for Disease Control and Prevention, U.S. , Humanos , Estados Unidos , Tos Ferina/epidemiología , Tos Ferina/inmunologíaRESUMEN
Cost-effectiveness analysis of an enzyme immunoassay (EIA) for the surveillance of arboviruses was conducted. The EIA was compared with conventional virus isolation and serologic identification procedures (virus isolation procedures; VIP). Under most circumstances, EIA was more cost-effective than VIP. Costs for processing mosquito pools by VIP increased with the number of viruses included in the surveillance program and with the prevalence rate of each virus. In contrast to VIP, the prevalence rate did not affect costs for processing pools by EIA. In general, EIA was the most cost-effective procedure, followed by cell culture and mouse bioassays. In a 5-year cost-effectiveness analysis of a model surveillance program in which EIA and cell culture bioassays were used, the EIA again proved to be the most cost-effective assay procedure under most circumstances.
Asunto(s)
Arbovirus/aislamiento & purificación , Culicidae/microbiología , Animales , Animales Lactantes , Análisis Costo-Beneficio , Técnicas para Inmunoenzimas , Ratones , ConejosRESUMEN
The envelope glycoprotein G, of human respiratory virus was purified by immunoaffinity chromatography using a monoclonal antibody reacting with G glycoprotein. The purified material was analyzed for its protein patterns and by western blot for its reactivity with specific monoclonal antibodies. In addition to the G specific proteins at 90 and 55 kilodalton (kDa) range, high molecular weight species were coeluted with G protein. Three high molecular weight species were noticed: one (140 kDa) reacting with fusion protein (F) monoclonal antibody and two other species (230 and 195 kDa) reacting with both fusion protein and G protein monoclonal antibodies. The protein reacting only with F monoclonal antibody consists of fusion protein dimer. Western blot and two dimensional gel electrophoretic analysis revealed that each of the other two complexes is composed of two moles of F protein and one mole of G protein. These two complexes differ in their molecular sizes depending on whether G is in the form of 90 or 55 kDa. Upon heat denaturation, fusion protein monomer (70 kDa) is released from the complex, leaving the two complexes, consisting of one mole of F protein and one mole of G protein (160 and 125 kDa species respectively). Disulfide-reducing agents are required to break the monomers of F and G complexes. These results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding. This may have implications on the structural and functional properties of envelope glycoproteins.
Asunto(s)
Antígenos Virales/metabolismo , Disulfuros/metabolismo , Proteína HN , Virus Sincitiales Respiratorios/metabolismo , Proteínas Virales , Anticuerpos Monoclonales , Antígenos Virales/aislamiento & purificación , Cromatografía de Afinidad , Electroforesis en Gel Bidimensional , Glicoproteínas/metabolismo , Calor , Immunoblotting , Oxidación-Reducción , Proteínas del Envoltorio ViralRESUMEN
The quaternary structure of respiratory syncytial virus (RSV) fusion protein has been studied. Crosslinking studies were done to stabilize the noncovalently associated proteins. These stable, heat-resistant, covalently linked complexes were analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis. In situ crosslinking studies demonstrated that the fusion protein of RSV exists as a dimer in its native form on the surface of infected cells. The purified protein was also found to be present predominantly as a dimer. In addition, the results suggest that F1 subunits may play a role in the dimerization of the fusion protein.
Asunto(s)
Antígenos Virales , Proteína HN , Virus Sincitiales Respiratorios , Proteínas Virales , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Conformación Proteica , Succinimidas , Proteínas del Envoltorio ViralRESUMEN
A method is given for estimating the infection rate in a population of organisms when variably sized sample pools are analyzed, a common situation in practice but not one which can be dealt with by existing methodology. An example is given of estimating the infection rate of yellow fever virus in a mosquito population; there is a suggestion that larvae with a longer developmental period had a higher infection rate. The minimum infection rate (MIR) parameter is shown previously for studies with a constant pool size; the MIR is however an acceptable estimate if the true infection rate is small.
Asunto(s)
Biometría/métodos , Métodos Epidemiológicos , Insectos Vectores/microbiología , Aedes/microbiología , Animales , Larva/microbiología , Virus de la Fiebre Amarilla/aislamiento & purificaciónRESUMEN
The region of the fusion glycoprotein of respiratory syncytial virus which reacts with a neutralizing and fusion inhibiting monoclonal antibody, was mapped using a deductive method derived from analysis of Western blot reactivity of proteolytic fragments. Reaction of the whole fusion protein was found to be so conformationally dependent, that complete digestion of the protein with a variety of proteases resulted in fragments which were not sufficiently reactive to permit mapping. For this reason, polyclonal antibodies to synthetic peptides which spanned the fusion protein sequence, were used to map the position of large peptides derived from partial digests, and these peptides were then analysed for their ability to react with the monoclonal antibody. Comparison of the peptides which were reactive with the monoclonal antibody to those which were not, identified a region of non-overlap between residues 283 and 327 in the F1 subunit of the fusion protein. Synthesis of a peptide within this region confirmed the placement of the epitope.
Asunto(s)
Antígenos Virales/química , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales de Fusión/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Endopeptidasas , Epítopos/análisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Mapeo Peptídico , Proteínas Virales de Fusión/químicaRESUMEN
Vaccination with a respiratory syncytial virus (RSV) fusion protein subunit vaccine (PFP-2) was done to determine if this vaccine induced evidence of cell-mediated immunity to RSV and if cell-mediated immunity prevented RSV reinfection. Healthy children 12-18 months old received 50 micrograms of PFP-2 or a saline control. Lymphocyte transformation (LTF) responses were determined before and 1 and 6 months after vaccination. PFP-2 induced positive LTF responses in 5 (83%) of 6 subjects whose prevaccination samples lacked evidence of cell-mediated immunity. Positive LTF responses in prevaccination samples were not boosted but were more persistent in vaccinees than in controls. Positive LTF responses were not associated with protection against subsequent infection. Immunization with PFP-2 induces correlates of cell-mediated immunity, but this immunity does not appear to be a critical component of protection.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Humano/inmunología , Vacunas Virales/inmunología , Método Doble Ciego , Humanos , Inmunidad Celular , Lactante , Activación de Linfocitos , Vacunación , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales de Fusión/inmunologíaRESUMEN
In a previous study, children 18 to 36 months of age and seropositive for respiratory syncytial virus (RSV) were vaccinated with an RSV subunit vaccine (PFP-1) consisting of the viral fusion protein. Vaccines developed substantial increases in anti-fusion and neutralizing antibody and exhibited protection against RSV infection through one RSV epidemic, in comparison to controls. This present study of the same cohort was undertaken to determine the persistence of antibody responses and immunity to reinfection, as well as to monitor for enhanced disease upon subsequent RSV infection during the second RSV season after vaccination. Vaccinees continued to have greater ELISA specific anti-fusion (F) antibody responses than controls up to 18 months after vaccination. Neutralizing antibody titres were not as durable, and the attack rates for RSV in the second winter season after vaccination (25% in vaccines versus 42% in controls) were not significantly different (p = 0.23). Nevertheless, 'high-responder' subgroups may have had residual protection into the second postvaccination year. Enhanced illness did not occur. PFP-1 is immunogenic and appears safe, but yearly reimmunization may be necessary to maintain immunity to RSV infection.
Asunto(s)
Anticuerpos Antivirales/sangre , Proteína HN , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Preescolar , Método Doble Ciego , Femenino , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina E/sangre , Lactante , Masculino , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Factores de Tiempo , Proteínas del Envoltorio ViralRESUMEN
We describe the covalent attachment of palmitate to the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site. Labeling of respiratory syncytial virus-infected Vero cells with [3H]palmitate, followed by the purification and subsequent analysis of the fusion glycoprotein in conjunction with polyacrylamide gel electrophoresis, demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein. The bound palmitate was sensitive to 1 M hydroxylamine at neutral pH. In addition, the release of palmitate label by reduction with sodium borohydride showed that the palmitate is linked to the protein through a thioester bond. Isolation of a radiolabeled peptide from a tryptic digest of the protein and subsequent amino-terminal sequence analysis revealed that the cysteine residue (amino acid residue 550) within the anchor sequence, located at the carboxyl terminus of the F1 subunit, is the covalent attachment site for palmitate.
Asunto(s)
Antígenos Virales , Proteína HN , Ácidos Palmíticos/metabolismo , Virus Sincitiales Respiratorios/metabolismo , Proteínas Virales , Acilación , Secuencia de Aminoácidos , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Datos de Secuencia Molecular , Ácido Palmítico , Fragmentos de Péptidos/aislamiento & purificación , Virus Sincitiales Respiratorios/inmunología , Proteínas del Envoltorio ViralRESUMEN
We studied the antibody response to the fusion (F) and attachment (G) proteins of respiratory syncytial virus and to purified intact virus in the respiratory secretions of 29 infants and children. The goal of the study was to determine whether the immune response to either of the glycoproteins occurred predominantly in the immunoglobulin A (IgA) as opposed to the IgE isotype, which would indicate that one protein subunit would be a better candidate as a potential vaccine. Antibody responses were determined by using an enzyme-linked immunosorbent assay with purified F and G proteins and sucrose gradient-purified intact virus as targets. Infants and children were capable of developing an antibody response in both the IgA and IgE isotypes to each target antigen. The magnitude of the antibody response to the F protein was essentially similar to that to the intact virus, while responses to the G protein were diminished in infants. A slightly more favorable ratio of IgA to IgE responses was observed against the F protein in comparison to the G protein. While neither protein subunit had the ideal characteristics of inducing an IgA response in the absence of an IgE response, the F protein seems to be a better candidate for use as a vaccine, on the basis of better IgA/IgE ratios.
Asunto(s)
Anticuerpos Antivirales/análisis , Inmunoglobulina A/análisis , Inmunoglobulina E/análisis , Isotipos de Inmunoglobulinas/análisis , Virus Sincitiales Respiratorios/inmunología , Infecciones por Respirovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Humanos , Lactante , Recién Nacido , Proteínas Virales de Fusión/inmunologíaRESUMEN
Hemagglutination titers of La Crosse virus antigens prepared in BHK-21 suspension cell cultures were substantially increased by precipitating the antigens with polyethylene glycol.
Asunto(s)
Bunyaviridae/inmunología , Virus de la Encefalitis de California/inmunología , Hemaglutininas Virales/aislamiento & purificación , Animales , Línea Celular , Precipitación Química , Cricetinae , Virus de la Encefalitis de California/crecimiento & desarrollo , Métodos , PolietilenglicolesRESUMEN
Aerosolized ribavirin was evaluated in the treatment of respiratory syncytial virus lower respiratory tract disease in 53 infants, 36 of whom had underlying diseases. Of the total infants, 26 were studied in a double-blind, placebo-controlled manner; 14 received ribavirin and 12 received placebo, a water aerosol, for an average of five days. When the infants with bronchopulmonary dysplasia and congenital heart disease treated with ribavirin were compared with those receiving placebo, the treated infants showed a significantly faster rate of improvement in their illness severity score. The degree of improvement in the total group of infants receiving ribavirin compared with those receiving placebo was similarly greater, and at the end of therapy significantly greater improvement was also demonstrated in their arterial blood gas values and in the amount of virus shed from their nasal washes. No toxic or adverse effects of the aerosol therapy were observed in any of the 53 infants studied, and resistance to ribavirin did not develop in any of the respiratory syncytial virus strains isolated, despite prolonged treatment in some of the more ill infants.