Comparative studies on the immunological response to the live spore anthrax vaccine in goats and passive protection test in mice

Abstract

Anthrax caused by the spore-forming bacterium Bacillus anthracis is primarily a disease of herbivorous animals and to a lesser extent in humans. Anthrax commonly infects wild and domestic ruminants that ingest or inhale the spores. Bacillus anthracis has several major virulence factors encoded on plasmids (pXO1 and pXO2), including the toxin components namely lethal factor (LF), edema factor (EF) and protective antigen (PA). The three exotoxin components combine to form two binary toxins. LF in combination with PA gives rise to lethal toxin (LT) which is lethal in several animal models including mice and guinea-pigs, and EF combines with PA to give oedema toxin (ET). Both LT and ET are responsible for the characteristic signs and symptoms of anthrax. Since PA complexes with EF and LF and interacts with host cell receptors to translocate the toxins into the cytosol, it plays an essential role in anthrax pathogenesis. Presently, the anthrax veterinary vaccine comprises of the live, attenuated B. anthracis 34F2 spores that was developed in 1937 by Max Sterne and known as the Sterne vaccine. The vaccine is a stable uncapsulated mutant that produces all three toxin components of B. anthracis (PA, LF and EF), but lacks plasmid pXO2 encoding capsule formation whilst still providing protection against B. anthracis infections. The Sterne vaccine is the most widely used veterinary vaccine, which provides safe and effective protection against anthrax in animals, by inducing immune response against antigens of the spore, cell mediated response and the toxins including protective antigen (PA) of B. anthracis, a component of anthrax toxin. While being effective in controlling anthrax globally, the live spore vaccine requires the use of animals for safety and efficacy tests. A major obstacle to improved anthrax vaccine research has been the lack of a convenient and sensitive method of monitoring the antibody response induced when the vaccine was developed. In a previous challenge study, revaccinated Sterne goats were 100% protected against virulent B. anthracis challenge but due to the lack of biosafety facility, we investigated the use of passive protection test in mice to evaluate anthrax vaccine protection. The main focus of the study was to develop a model that correlates immune response induced by the live spore anthrax vaccine in goats with survival through the passive protection test in mice. Furthermore to investigate the performance of macrophage cell lines from target host in an existing toxin neutralization assay against the standard mice macrophage cell line in same assay. Five Boer goats were vaccinated twice over 3 months (week 0 and week 12) with 1ml of the Sterne live spore vaccine (at spore concentration of 6 x 106 per dose) and a negative group (n=3) consisted of naive goats received only sterile saline (1 ml) administered subcutaneously. Enzyme-linked immunosorbent assay (ELISA) and anthrax toxin neutralization assays (TNA) were used to monitor the anti-PA and toxin neutralizing antibodies of vaccinated and non-vaccinated (naive) goat groups after two vaccinations (week 4 and week 17). The anti-PA ELISA antibody titres indicated no significant difference between unvaccinated and goats vaccinated one (week 0 and 4) whereas a significant difference in the titer could be seen after the second vaccination (week 0 and 17). The TNA titres after the first vaccination (week 4) were low and with two goats were nil. All goats developed toxin neutralizing titres after the second vaccination (week 17). The ELISA and TNA assay titres showed no correlation of vaccinated animals at 17 weeks after the initial vaccination (r2 = -0.177). The TNA was done using mice macrophage cell line (BALB/c monocyte macrophage cell line J774A.1). The TNA assay could not be done using caprine, bovine (BOMAC) and canine (DH 82) macrophage cell lines as a continuous caprine macrophage cell line could not be obtained and the toxins had no effect on the bovine (BOMAC, host) and canine (DH 82, non-host) macrophage cell lines.