ABSTRACT
Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.
Subject(s)
Anthrax/blood , Anthrax/diagnosis , Bacillus anthracis/genetics , Bacillus anthracis/isolation & purification , Bioterrorism , Oligonucleotide Array Sequence Analysis/methods , Polymerase Chain Reaction , Animals , Anthrax/microbiology , MiceABSTRACT
The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2) recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete protection was achieved when the proteins were given alone or in combination with DNA vaccines. These data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea pigs.