BclA and toxin antigens augment each other to protect NMRI mice from lethal Bacillus anthracis challenge

Highlights

• Combining toxin and spore antigens results in 70–90% survival after lethal challenge.

• Anti-spore and anti-toxin antigens augment each other in DNA and protein form alike.

• DNA-vaccines combining epitopes of LF, PA and BclA elicited 90% protection.

• BclAD1D3 as single component elicits 50% protection and potentially sterile immunity.

• Antibodies against LFD1PAD4 have neutralising qualities.

Abstract

While proving highly effective in controlling Anthrax in farm animals all over the world currently attenuated live anthrax vaccines employed in a veterinary context suffer from drawbacks such as residual virulence, short term protection, variation in quality and, most importantly, lack of efficacy if administered simultaneously with antibiotics. These limitations have stimulated the development of non-living component vaccines which induce a broad spectrum immune response capable of targeting both toxaemia (as in the case of PA based vaccines) and bacteraemia. To contribute to this several new approaches were tested in outbred NMRI mice for antibody titres and protectiveness. Plasmids encoding a recombinant toxin derived fusion peptide and a spore surface derived peptide were tested as DNA-vaccines in comparison to their protein counterparts utilising two adjuvant approaches and two DNA-vector backbones. The combination of two plasmids encoding LFD1PAD4-mIPS1 and TPA-BclAD1D3-LAMP1, when delivered by GeneGun, protected 90% of the animals against a lethal challenge with 25LD₅₀ spores of the Ames strain of Bacillus anthracis. Single applications of either antigen component showed significantly lower protection rates, indicating the beneficial interaction between anti-spore and anti-toxin components for an acellular vaccine formulation.

Introduction

Anthrax is caused by Bacillus anthracis, a Gram-positive, spore forming, rod-shaped bacterium [1]. Spores gain access via cutaneous, oral or inhalational routes where they germinate and develop into vegetative bacilli which then replicate and produce toxins which eventually kill the host [2]. The pathogen expresses two major plasmid encoded virulence factors, a gamma-linked poly-d-glutamic acid capsule (pX02 [3]) and a tripartite toxin (pX01 [4]) comprised of Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF) [5], [6].

Current live attenuated veterinary anthrax vaccines are less than ideal. They can cause problems in sensitive animals such as goats and llamas, protection is short term, variation in vaccine quality can cause vaccine failure and most importantly the live nature of the vaccine prevents its efficacy if delivered at the same time as antibiotics [7], [8]. These limitations have stimulated the development of non-living, component vaccines capable of inducing a broad spectrum immune response which targets both toxaemia and bacteraemia.

The strong correlation between toxin neutralising activity (tna) of PA-specific antibodies and protection [9] has prompted efforts to develop vaccines based solely on domains which stimulate antibodies with tna [10], [11], [12]. One such study which employed a fusion protein comprised of domain 4 of PA (receptor binding site) and domain 1 of LF (PA binding site) protected mice against a subsequent lethal challenge with B. anthracis spores [13]. To further assess the immunogenic value of this protein we administered it as a DNA-vaccine in two different vectors and compared its activity to that seen against full length rPA83.

In addition to neutralising the action of toxins the spore can also be targeted to prevent the pathogen from gaining a foothold in the infected individual [14], [15]. Vaccination experiments with live nonvirulent or formaldehyde-inactivated spores have shown that spore specific immune responses can enhance the level of protection when given in combination with PA [16].

One such component is the Bacillus collagen like protein of anthracis (BclA) which forms hair-like structures projecting from the spore surface and represents a major spore immunogen [17], [18]. The removal of the collagen-like region (CLR, domain 2) from BclA has no detrimental effect on immunogenicity and results in a smaller peptide which is easier to incorporate into a multicomponent vaccine [19], [20]. In this study we determined the immunogenicity of a CLR-deficient version of BclA called rBclAD1D3 when administered as a DNA-vaccine in two different vectors.

For the DNA vaccine studies we employed two different plasmid backbones (pDNAVaccUltra and NTC7382) which varied with regards to intracellular routing signals and immune stimulatory elements [21]. To improve in vivo antigen presentation we utilised intracellular routing signals which directed vaccine peptides to the MHC I and MHC II pathways. To target the MHC II pathway [22] we employed tissue plasminogen activator (TPA) which routes newly expressed proteins to the secretion pathway [23] and lysosome-associated membrane protein (LAMP1) which directs proteins to the endosome [24], [25]. To enhance MHC I presentation we employed ubiquitin which directs the associated protein to the proteasome [26], [27].

To enhance the immunogenicity of the expressed proteins we investigated the utility of two molecular adjuvants. Mouse interferon-ß promoter stimulator 1 (mIPS-1) incorporated into the backbone of the antigen encoding plasmid significantly induces type I interferon and interferon-stimulated genes in a TLR-independent matter [28], [29], [30]. Mouse class II MHC trans-activator (CIITA) up-regulates MHC expression [31], [32] and was co-administered on a separate plasmid.

In comparison to the DNA vaccines, full length rPA and rBclA were tested as proteins alone and in combination in the presence of a previously tested and approved lipopeptide adjuvant comprising Pam3Cys-SKKKK, a TLR2/1 activator admixed with Pam₃Cys conjugated to the promiscuitive T-helper-cell epitope of the sperm whale myoglobin SFISEAIIHVLHSRHPG [33], [34].

The overall aim of this study was to determine the ability of BclA to confer additional protectiveness when given together with a toxin-specific vaccine.