Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model
Introduction
Anthrax is a primary disease of herbivores caused by the Gram-positive bacterium Bacillus anthracis (Hambleton et al., 1984, 125–132). The disease develops as a peracute or acute infection in ruminants following an incubation period of 3–5 days (Beyer and Turnbull, 2009, 481–489), which often elapses without clinical signs until shortly before death due to sudden septicemic shock. Clinical signs and course of infection are largely dependent on the species and the route of infection with ambiguous early signs (Hambleton et al., 1984, 125–132). The pathogen expresses two major plasmid encoded virulence factors, a gamma-linked poly-d-glutamic acid capsule [(PGDA) (coded by pX02)] and a tripartite toxin (coded by pX01) comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) (See review Mourez, 2005, 135–164). Virulence of B. anthracis is dependent on the presence of both plasmids (Green et al., 1985, 291–297).
The PDGA is weakly immunogenic and assists in post infection dissemination of B. anthracis (Candela and Fouet, 2005, 717–726). The capsule enables the anthrax bacilli to evade immune surveillance mechanisms and enter the circulatory system where it proliferates systemically (Sutherland et al., 2008, 899–906). PA combines with LF to form lethal toxin, a zinc metalloprotease that inactivates most mitogen-activated protein kinase kinases (MAPKK) and the inflammasome-activating NLRP1B leading to impairment and death of susceptible macrophages (Friedlander, 1986, 7123–7126; Chavarría-Smith and Vance, 2013, e1003452). Edema toxin (ET), a calmodulin dependent adenylate cyclase formed by the binding of PA to EF, disrupts fluid homeostasis across the host cell membranes (Mourez, 2005, 135–164).
The current anthrax veterinary Sterne live spore vaccine (SLSV) is a non-encapsulated but toxigenic variant 34F2 that was developed in 1937 by Max Sterne (Sterne, 1937, 49–67). This vaccine is still extensively used in the control of anthrax in domestic animals (International Office of Epizootics, 2008, 94–95). Constraints that include limited duration of immunity, failure to induce protective immunity, variation in vaccine quality and adverse reactions in sensitive species, such as llamas (Lama glama) and goats (Capra aegagrus hircus) have been reported (Cartwright et al., 1987, 715–716, Turnbull, 1991, 533–539). The current OIE manual stipulates annual vaccination of animals against the disease (OIE, 2012, 135–144). Immune parameters and correlates to protection for anthrax in goats are limited in literature because serological methods were not readily available during earlier vaccine trials in domestic species. These trials mainly depended on clinical responses and level of protection after vaccination (Sterne, 1937, 49–67; Sterne et al., 1942, 53).
The principal immune response induced by vaccination of animals with the SLSV is the development of antibodies against PA (International Office of Epizootics, 2008, 94–95). Anti-PA antibodies prevent the development of lethal intoxication and protection against anthrax is mainly provided by the development of immunity to the antigen (Shakya et al., 2007, 5374–5377). The presence of antibodies against the spore (formaldehyde inactivated spores, FIS) and spore-associated antigens such as the bacillus collagen-like protein of anthracis (BclA) and vegetative antigens has been reported to augment the protection afforded to animals (Brossier et al., 2002, 661–664; Hahn et al., 2006, 4569–4571). Hence we sought to evaluate the humoral immune response in Boer goats directed against these antigens following single or booster vaccinations with the SLSV. Antibody responses were assessed using ELISA. Also, the ability of induced antibodies to neutralize lethal toxin was measured using the in vitro toxin neutralization assay (TNA). The level of protection following vaccination was evaluated by challenge with virulent B. anthracis spores.
Section snippets
Animals
Eight-week old female BALB/c mice [(n = 6) (South African Vaccine Producers, Sandringham, South Africa)] were used to confirm the virulence of the B. anthracis challenge strain (Welkos et al., 1986, 795–800). Twenty-six 1-year naïve old Boer goats were housed at Onderstepoort Biological Products (OBP), South Africa after screening for anti-rPA83 cross-reactive antibodies. Lethal challenge studies were conducted at a remote site in an endemic area of the Kruger National Park (KNP), South Africa.
Recombinant protective antigen (rPA83) ELISA and toxin neutralizing assay (TNA) titres
A single vaccination with the SLSV induced high anti-rPA83 IgG and toxin neutralizing titres within four weeks (Fig. 1, Fig. 2). After the initial recorded peak, the titres declined to a constant level that was still significantly elevated (P ≤ 0.017) until either challenge (SVG2) or revaccination (SVG3) when compared to titres before vaccination. Revaccination at week 58 with the SLSV (SVG3) induced higher rPA83 and toxin neutralizing antibodies compared to SVG2 titres as can be seen in Fig. 1,
Discussion
In the early 20th century, anthrax was a major cause of death in livestock within South Africa [(over 1800 outbreaks reported in 1920) (Gilfoyle, 2006, 465–490)]. The introduction of compulsory vaccination of livestock with the SLSV drastically reduced the disease incidence. However, little is known about the specific immunity induced by the vaccine in the target ruminant host. It should be noted that most of the extensive research done on anthrax vaccines were performed on laboratory rodents
Funding
This study was funded by the German Research Foundation (DFG) with grant # BE2157/4-1.
Acknowledgements
We are hugely indebted to Peter Turnbull for his invaluable technical advice on this project, the staff of OBP and the Veterinary Service, Directorate of Animal Health, Department of Agriculture, Forestry and Fisheries (DAFF), Skukuza State Veterinary Office (especially Dr Roy Bengis) and SANParks for taking care of the animals and assistance with the animal experiments.
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- 1
These authors contributed equally to this work.
- 2
Current address: Robert Koch Institute, Nordufer 20, 13353, Berlin, Germany.