RESUMEN
Identification of neutralizing antibodies with specificity away from the traditional mutation prone antigenic regions, against the conserved regions of hemagglutinin from H5N1 influenza virus has the potential to provide a therapeutic option which can be developed ahead of time in preparation for a possible pandemic due to H5N1 viruses. In this study, we used a combination of panning strategies against the hemagglutinin (HA) of several antigenic distinct H5N1 isolates to bias selection of Fab-phage from a naïve human library away from the antigenic regions of HA, toward the more conserved portions of the protein. All of the identified Fab clones which showed binding to multiple antigenically distinct HA were converted to fully human IgG, and tested for their ability to neutralize the uptake of H5N1-virus like particles (VLP) into MDCK cells. Five of the antibodies which showed binding to the relatively conserved HA2 subunit of HA, exhibited neutralization of H5N1-VLP uptake in a dose dependant manner. The inhibitory effects of these five antibodies were similar to those observed with a previously described neutralizing antibody specific for the 140s antigenic loop present within HA1 and highlight the exciting possibility that these antibodies may be efficacious against multiple H5N1 strains.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Hemaglutininas Virales/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Línea Celular , Perros , Regulación Viral de la Expresión Génica , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Internalización del VirusRESUMEN
The monoclonal antibody VN04-2 was previously shown to protect mice against lethal A/Vietnam/1203/04 H5N1 virus challenge when administered pre- and post-infection. In this study, we characterized the binding requirements of this antibody using direct binding to hemagglutinin and neutralization assays with H5N1 virus-like particles (H5N1-VLP) of eight recent H5N1 strains representing the major mutations within the 140s antigenic loop. Binding was clade independent and 3 mutations within this antigenic region are required before escape is possible, suggesting that apart from the H5N1 viruses circulating in Indonesia, VN04-2 may provide protection against H5N1 viruses from all other regions.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Mutación , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Epítopos/química , Epítopos/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Indonesia , Subtipo H5N1 del Virus de la Influenza A/clasificación , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Ratones , Pruebas de Neutralización , Virión/inmunologíaRESUMEN
Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.
Asunto(s)
Anticuerpos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Proteínas/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Proteínas/genética , Reproducibilidad de los Resultados , Anticuerpos de Cadena Única/genéticaRESUMEN
The present study examined the effectiveness of a humor therapy program in relieving chronic pain, enhancing happiness and life satisfaction, and reducing loneliness among older persons with chronic pain. It was a quasiexperimental pretest-posttest controlled design. Older persons in a nursing home were invited to join an 8-week humor therapy program (experimental group), while those in another nursing home were treated as a control group and were not offered the program. There were 36 older people in the experimental group and 34 in the control group. Upon completion of the humor therapy program, there were significant decreases in pain and perception of loneliness, and significant increases in happiness and life satisfaction for the experimental group, but not for the control group. The use of humor therapy appears to be an effective nonpharmacological intervention. Nurses and other healthcare professionals could incorporate humor in caring for their patients.