Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax.

Bacillus anthracis is the causative organism of the disease anthrax. The ability of the organism to form resistant spores and infect via the aerosol route has led to it being considered as a potential biological warfare agent. The current available human vaccines are far from ideal, they are expensive to produce, require repeated doses and may invoke transient side-effects in some individuals. There is also evidence to suggest that they may not give full protection against all strains of B. anthracis. A new generation of anthrax vaccine is therefore needed. The use of Lactobacillus as a vector for expression of heterologous proteins from pathogens supplies us with a safe system, which can be given orally. Lactobacilli are commensals of the gut, generally regarded as safe and have intrinsic adjuvanticity. Oral vaccines may stimulate the mucosol immune system to produce local IgA responses in addition to systemic responses. These vectors are delivered at the mucosal surface, the site where the infection actually occurs and where the first line of defence lies. The gene encoding the protective antigen (PA) of B. anthracis, an immunogenic non-toxic component of the two toxins produced, is being cloned into different homologous vectors and subsequently transformed to various Lactobacillus strains. High intracellular expression levels for the PA in Lact. casei were achieved. Mucosal antigen presentation and humoral and cellular immune responses following immunization with transformants expressing PA in various ways (intracellular, surface-anchored and extracellular) are being studied.

Rapid in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies to both viruses and toxins.

A generally applicable method was developed for the rapid and quantitative detection of both toxin- and virus neutralizing antibodies. The method was optimized for three different biological agents, i.e., Shigella toxin, influenza viruses (A/Beying, A/Taiwan and B/Yamagata) and Chikungunya virus. The in vitro micro-cytotoxicity tests developed for the detection and quantitation of neutralizing antibodies are based on the inhibition of the virus- or toxin-induced cytotoxic effect by antibodies. As a result of the cytotoxicity, infected cells are no longer attached to the solid phase and can be easily removed. Thereafter, the proteins of the remaining living cells are stained. After removing the excess dye, the remaining dye is dissolved and the absorbance values are measured. The neutralization titers are determined from the absorbance values. Since the tests are performed in wells of microtiter plates, the in vitro micro-cytotoxicity tests are less laborious and consume less reagent in comparison with classical neutralization tests.

Changes in circulatory white blood cells of mice and rats due to acute trichothecene intoxication.

In mice, administration of pure T-2 toxin caused a rapid decrease of lymphocyte counts, which was linear with respect to dose, whereas granulocyte counts showed a delayed decrease. The blood cell counts of both cell types attained normal values after 4-7 days. Similar results were obtained for crude A-, B- and macrocyclic type trichothecene. Intoxication of rats with T-2 toxin or crude A-type trichothecene caused changes in white blood cells, which differed quantitatively from those in the mouse: lymphocyte counts decreased less and a rapid transient increase of granulocytes was more obvious. Results of this study show that lymphocyte and granulocyte blood cell counts of small rodents respond sensitively to acute intoxication with various trichothecenes.