ABSTRACT
Western equine encephalitis virus (WEEV) is a mosquito-borne RNA virus which causes lethal infection in humans and equines. There are no commercial vaccines or anti-WEEV drugs available for humans. We used replication-defective, human adenovirus serotype-5 (HAd5) as a delivery vector for developing WEEV vaccine. Our previous study found delivery of both E1 and E2 envelope proteins of WEEV by HAd5 vector offers complete protection against lethal challenge of WEEV. In this paper, we constructed a HAd5-vectored E1 vaccine, Ad5-E1. Mice given single-dose vaccination of Ad5-E1 were completely protected against both homologous and heterologous WEEV strains. The protection was rapid, which was achieved as early as day 7 after vaccination. In addition, Ad5-E1 induced a strong WEEV-specific T cell response. Our data suggest E1 is a potential target for developing single-dose, fast-acting, HAd5-vectored vaccine for WEEV.
Subject(s)
Encephalitis Virus, Western Equine/immunology , Encephalomyelitis, Equine/prevention & control , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Adenoviruses, Human/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Disease Models, Animal , Drug Carriers , Encephalomyelitis, Equine/immunology , Female , Genetic Vectors , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Survival Analysis , T-Lymphocytes/immunology , Viral Plaque AssayABSTRACT
A microsphere-based suspension array (SA) system was used for the development and characterization of an immunoassay for the toxin simulant ovalbumin. Results obtained by SA immunoassay were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) using the same immunoreagents. The limit of detection (LOD) for the SA ovalbumin assay was 4.9 ng/mL, compared to a LOD of 0.01 ng/mL for the ovalbumin ELISA. Although the ELISA LOD exceeded that of the SA assay, the SA assay was simple and rapid to perform, with assays being completed in half the time of the traditional ELISA. The well-to-well reproducibility (coefficient of variation (CV)) of the ELISA and the SA assay was 4.9% and 5.1%, respectively. The ELISA and SA assay plate-to-plate reproducibility was 14.8% and 6.1%, respectively. The protocols used to develop the SA assay for ovalbumin may be used as a template for development of other SA assays for toxins, bacteria, and viruses.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Ovalbumin/analysis , Protein Array Analysis/methods , Toxins, Biological/analysis , Animals , Antibodies, Monoclonal/immunology , Mice , Microspheres , Reproducibility of Results , Sensitivity and Specificity , SuspensionsABSTRACT
In the process of generating the cells that populate the sheep's B-cell pool, the ileal Peyer's patch (PP) produces an immense number of B-cells and then destroys most of them by apoptosis. Rapid clearance of these apoptotic cells is essential for tissue homeostasis and for preventing pathology. Macrophages comprise a small percentage of cells in the follicles. They resemble macrophages found in other tissues and can be identified by the expression of MHC Class II and CD14. In this study, enriched macrophages co-cultured with apoptotic ileal PP cells showed increased DNA content as they ingested apoptotic cells. The higher the proportion of apoptotic cells in culture the greater the increase in DNA content of the macrophages. This occurred when B-cell apoptosis was initiated by a period in culture or in response to treating the animals with steroids. Thus, macrophages resident in the ileal PP follicle mediate the phagocytosis and removal of discarded B-cells.