CBMTS IV: Selected Abstracts

Anthrax Vaccines: Future Targets
Robert M. DeBell, Ph.D. , Battelle Memorial Institute

          In 1997, a Russian paper in the journal Vaccine described the transfer of the cereolysin AB genes from B. cereus to B. anthracis. This recombinant B. anthracis lysed red blood cells and caused anthrax in Golden hamsters vaccinated with the STI-1 vaccine. These two observations may not be mutually exclusive. A recent report from a group at the Institut Pasteur showed that the insertion of a multicopy plasmid containing the plcR gene into B. anthracis resulted in the expression of several proteins. Similar results were obtained when a single copy of the plcR gene was inserted into the chromosome of B. anthracis. In both cases, the recombinant organisms showed hemolytic activity. Using an E. coli T7 expression system, another research group cloned the cereolysin AB genes (cerA and cerB) and the plcR gene from both B. anthracis and B. cereus. They found that cerA and cerB in B. anthracis are functionally active and similar to those of B. cereus.
          Although B. anthracis contains a shortened, somewhat dysfunctional plcR gene, these studies suggest that B. anthracis contains several genes that can be controlled by PlcR. Based on the studies described, it may be possible that the expression of the otherwise silent B. anthracis cereolysin genes may alter the typical anthrax disease process. To ensure that anthrax vaccines are effective, it may be important to change or expand the target molecules beyond protective antigen used in both the U.S. and U.K. vaccines. In the 1997 Vaccine paper, hamsters vaccinated with a recombinant (i.e., STI-1 with cereolysin AB genes inserted) vaccine strain were protected against the recombinant pathogen. Future molecular targets for vaccines may include cereolysins AB and possibly PlcR.

Keywords: vaccines, anthrax, cereolysins, hemolytic activity

Studies of Variola Virus Replication in Cell Cultures and Chick Embryo Chorion-Allantoic Membrane
Elena Ryabchikova, et al
State Research Center of Virology and Biotechnology "Vector", Russian Ministry of Public Health.

          Reproduction of variola virus (strains India-3, Congo-9 and Butler) having different geographic origination has been examined in cell cultures originated from primates (Vero and CV-1), humans (Fl and L-68) and hamster (BHK-21). The production of extracellular and intracellular virus was determined by biotitration on chick embryo chorion-allantoic membrane. Characteristics of virus assembly in cultured cells were examined by electron microscopy. Differences in the pattern of reproduction of Variola virus strains in the listed cell cultures have been described.
          Morphology and virus reproduction have been examined in pocks caused by Variola virus (strains India-3, Congo-9 and Butler) on chick embryo chorion-allantoic membranes. This experimental model provides for examination of the virus infection on the background of "immature" immunity, and represents a model of "mucous membrane lesion". The results showed clear differences in the local inflammatory reaction induced by different viral strains. The most prominent damage of tissues was caused by the India-3 strain of variola virus. Features of the reproduction of Variola virus strains in cell cultures and chick embryo cells are analyzed and compared.

Keywords: Variola virus, pox viruses, electron microscopy

Overview of the US National Pharmaceutical Stockpile Program; Providing Technical Assistance Through Training, Education And Demonstration
Steven D. Bice, National Pharmaceutical Stockpile Program

          The purpose of this presentation is a] to provide a brief overview of the U.S. National Pharmaceutical Stockpile Program; and, b] to discuss how the NPS Program utilizes the Training, Education and Demonstration Packages to accomplish the task of familiarizing state and local responders with the Stockpile and how it is to be incorporated in their response efforts.
          The National Pharmaceutical Stockpile (NPS) represents a national repository of antibiotics, chemical antidotes, antitoxins, vaccines, life-support medications, IV administration and airway maintenance supplies and medical /surgical and trauma medications and equipment. The NPS Program is designed to supplement and re-supply U.S. state and local public health agencies and emergency first responders in the event of a terrorism incident anywhere in the US, at anytime. Working with state and local responders, public health specialists and emergency workers, staff of the NPS Program designed two training "packages" which are referred to as Training, Education and Demonstration (or TED) Packages for use in field exercises, static displays, at conferences/symposia, etc. This is the first year both "TED's" will be used around the US to help familiarize state and local responders with how the NPS is packaged, the specially designed cargo containers used, the load plans, the configuration of the NPS Push Packages and vendor managed inventory, the equipment, supplies and medical materiel which makes up the NPS.

Keywords: stockpile, Centers for Disease Control and Prevention, training, demonstration, pharmaceuticals

Cholinesterase Activity Assays: Molar Absorption Coefficient for the Reduced Ellman Reagent
Elsa Reiner, Franz Worek Institute for Medical Research and Occupational Health, Zagreb and Institute of Pharmacology and Toxicology, FAF Medical Academy, Munich

          The Ellman spectrophotometric method for assaying the hydrolysis of thiocholine esters by cholinesterases is based on the reaction of the chromogenic DTNB (5,5'-dithio-bis(2- nitrobenzoic acid)) with thiocholine whereby the rate of formation of the yellow 5-thio-2-nitrobenzoic acid anion (TNB) is measured. The TNB millimolar absorption coefficient as published by Ellman et al. (1959) is 13.6 at 412 nm, pH=8.0, temperature not stated. Over the years, slightly different values have been published, and it has further been shown that TNB absorption spectra are shifted to longer wavelengths when temperature is increased. Our coefficients measured at 412 nm in phosphate buffer pH=7.4 are 14.0 at 25 oC and 13.7 at 37 oC (measured with DTNB in excess over glutathione) and 14.2 at 25 oC (measured with cysteine in excess over DTNB). As the Ellman method is widely used, a critical review of published data seems required in order to apply values of the coefficient corresponding to experimental conditions used in a given cholinesterase activity assay.

Keywords: cholinesterase assays; reduced Ellman reagent absorption coefficient