Anthrax vaccination induced anti-lethal factor IgG: Fine specificity and neutralizing capacity

Abstract

The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-protective antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to lethal factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.

Introduction

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the causative agent of anthrax infection. Infection can be initiated by cutaneous, gastrointestinal, or inhalational routes, with the inhalational route resulting in 45–90% mortality [1]. Systemic infection is characterized by an extremely high blood concentration of bacilli, resulting in high concentrations of the secreted tripartite toxin. This toxin is composed of three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds to its cellular receptor(s), Tumor Endothelial Marker 8 (TEM8) or Capillary Morphogenesis Protein 2 (CMG2) [1], [2], [3], and is cleaved by a furin-like membrane endoprotease. The resulting 63 kDa fragment oligomerizes and, when endocytosed, carries EF and/or LF into the cell [1], [4], [5]. Edema toxin (ET), composed of PA and EF, is an adenylate cyclase that results in edema and can be lethal when injected into animals [6]. This toxin has also been shown to impair macrophage phagocytosis and increase cAMP levels [1], [7]. Lethal toxin (LT), formed by the combination of PA with LF, is a zinc-dependent protease that causes lysis of intoxicated macrophages and is lethal in animal models [1], [8]. Following PA-mediated translocation of LF into the cytosol, target cells such as macrophages release pro-inflammatory cytokines inducing endothelial cell death by apoptosis and leading to vascular collapse [1], [8], [9], [10], [11], [12], [13], [14].

The design of the current United States anthrax vaccine (Anthrax Vaccine Absorbed, AVA) is predicated on the fact that PA serves as a crucial component of both LT and ET, and antibodies against PA are known to provide protection from disease in animals [15], [16]. This vaccine is produced from a cell-free filtrate of an attenuated bovine isolate (V770-NP1-R) that produces a higher fraction of PA [17]. However, all three toxin components (PA, LF, and EF) are present in the product [17], [18]. While it is clear that antibodies to PA are the primary method of protection generated following AVA immunization, studies with mouse models have demonstrated the protective significance of antibodies to LF alone [19]. Antibodies directed against LF have been shown to provide protection against challenge with toxin or bacteria in several experimental animal models [14], [20], [21], [22], [23], [24], [25]. Additionally, the protective capacity of neutralizing antibodies directed against PA can be greatly enhanced by the addition of LF neutralizing antibodies [21]. Limited human data exists characterizing the fine-specificity and potential for protection of antibodies to LF following AVA immunization. This study evaluated plasma from a large cohort (n = 1000) of AVA immunized individuals for the quantitative levels of LF specific antibodies as well as for the presence of binding to sequential B cell epitopes that contribute to functional protection. Antibodies directed against two antigenic regions of LF, one in the PA binding domain and one in the ADP-ribotransferase-like domain, are able to provide protection in an in vivo mouse model of lethal toxin challenge. These data suggest that development of new active and passive vaccination strategies that incorporate these LF antigenic regions will lead to improved protection against anthrax.