Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

Abstract

dBacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 g/ml, with some as high as 10 mg/ml. The levels were 10-to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 g/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 g/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Bacillus anthracis produces two toxins, which are responsible for allowing the bacterium to establish disease and induce lethality in the host. Lethal toxin (LT) and edema toxin (ET) are composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is a receptor-binding component that transports LF (a protease) or EF (an adenylate cyclase) into cells where they can manifest their catalytic activities through the targeting of ubiquitous substrates. EF targets ATP and converts it to cyclic AMP (cAMP), resulting in cellular dysfunction and vascular events that can lead to lethality. LF cleaves the mitogen-activated protein kinase (MEK) family and rodent nucleotide-binding domain and leucine-rich repeat containing a pyrin domain 1 (NLRP1) inflammasome sensors. LF plays an important role in both early and late anthrax infection. Early in infection, inactivation of the MEK proteins by cleavage leads to the inhibition of a wide variety of innate immune cell responses, which allows the bacterium to evade the immune system, divide, and disseminate. The cleavage of NLRP1 early in infection in certain inbred rodents results in the activation of the inflammasome, macrophage pyroptosis, and induction of proinflammatory cytokines, which induce a protective immune response. Thus,...

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection

“…4 and 5). VHH-based agents are also more versatile as they express well as multifunctional fusion proteins (6-8, 18 -20) and can be delivered efficiently by gene therapy methods (15,21). A possible disadvantage of VHH-based agents for some applications is their shorter serum half-lives compared with conventional antibodies.…”

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking

“…While monomeric V H Hs generally have little toxin-neutralizing activity in vivo , bispecific (heterodimeric) V H H constructs consisting of two different V H Hs joined via a flexible peptide linker have proven to be remarkably effective at affording passive protection against extraordinarily high dose toxin challenges [ 14 , 20 ] or even, in the case of anthrax toxin, a spore challenge [ 19 ]. The prophylactic and therapeutic potential of these so-called VHH-based neutralizing agents (VNAs) is even more remarkable considering that they have been successfully engineered and administered to mice and piglets via a non-replicating adenovirus vector [ 14 , 21 , 22 ].…”

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