Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge

Abstract

We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.

Introduction

Botulinum neurotoxin (BoNT) is produced by the anaerobic soil organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum in seven distinct serotypes (serotypes A–G) [1], [2], [3]. BoNT toxin causes a serious and potentially life-threatening natural disease called botulism [1], however, the disease can occur as a result of bioterrorism and biological warfare [4]. BoNTs are produced as single-chain protoxins and activated by proteolytic cleavage into disulfide bond-linked di-chains consisting of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC). The 50-kDa LC contains a catalytic (zinc endopeptidase) domain that targets one of three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins of the synaptic vesicle-associated membrane protein synaptobrevin, synaptosomal-associated protein of 25 kDa, or plasma membrane associated SNARE syntaxin. Collectively, the SNARE proteins constitute the core of the vesicular fusion machinery enabling the fusion of neurotransmitter-containing synaptic vesicles with the neuronal plasma membrane, thus allowing the release of neurotransmitter into the synaptic cleft. Cleavage of one of these proteins prevents membrane fusion and release of neurotransmitter from the nerve cell, which results in the cessation of nerve impulses along the axon. The toxin HC contains the cell receptor-binding (fragment C or HC) and translocation (HN) domains necessary for the selective uptake of toxin into the nerve cell via receptor-mediated endocytosis and the subsequent trafficking of the endopeptidase across the endosomal membrane into the cytosol, where SNARE protein inactivation occurs [5], [6].

Inactivated rabies virus (RABV) has long been used to vaccinate humans with a significant history of safety [7], [8], and induction of potent and long-lasting neutralizing antibodies to the RABV glycoprotein (G) [9]. In addition, strong humoral responses to heterologous proteins, such as, HIV-1 envelope [10], [11]; HCV E1/E2 [12]; and anthrax PA [13] were elicited by inactivated RABV virions incorporating these foreign proteins. The highly repetitive and rigid organization of viruses and virus-like particles is thought to maximally stimulate B cells [14], [15]. Additionally, CD4⁺ T helper cell epitopes that are necessary for B cell activation can be provided by the virus-like particles [16].

Vaccination is an effective strategy for providing protection against botulinum toxin by eliciting neutralizing antibodies that would prevent binding of the toxin to an appropriate receptor and promote clearance and degradation by phagocytosis. Chemically detoxified botulinum vaccines have played an important role in providing active immunity against the neurotoxin [17], and for production of equine-based antitoxins and botulism immune globin for treating botulism by passive immunization [18], [19], [20], [21]. However, recombinant technology has provided the means by which highly purified and efficacious antigens can be produced in adequate quantities without the need to culture and manipulate large volumes of C. botulinum and its neurotoxin [22], [23], [24]. BoNT(HC) delivery has been reported using naked DNA [25], [26], virus vector system [27], [28], [29], [30], [31] and attenuated Salmonella typhimurium strains [32].

Here, we generated several chimeric RABV G-HC50/A fusion proteins containing the RABV G ER translocation sequence (SS), various RABV ectodomain (ED) fragments (5–51 aa) and the RABV transmembrane TM and cytoplasmic domain (CD). HC50/A chimeric protein was expressed at the highest level on the surface infected BSR cells when a 30 aa fragment of the RABV ED was utilized, which resulted in efficient incorporation into RABV virions, as well. Mice vaccinated with inactivated RABV-HC50/A-E30 virions mounted neutralizing antibody response against BoNT even when low amounts of the vaccine was used. In addition, vaccination with inactivated RABV-HC50/A-E30 virions led to rapid clearance of the toxin from the circulation and elicited protective immunity in mice against lethal BoNT/A challenge. These results provide further evidence the potent immunostimulatory potential of inactivated RABV, which can serve as a new carrier for immunization against various pathogens.