Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system

Abstract

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

FIG. 1.

In vitro expression of HcA using linear template DNA. (A) Two-step PCR was used to add all the regulatory elements required for efficient in vitro transcription and translation of HcA. The first PCR generated universal sequences that complemented the 2nd PCR primers in which regulatory elements were added. The PCR products of the first (lanes 2 and 3) and second (lanes 4 and 5) PCRs for HcA-His₆ (lanes 2 and 4) and His₆-HcA (lanes 3 and 5) were separated on 1% agarose gel. (B) SDS-PAGE and immunoblotting of the in vitro-expressed proteins. The recombinant proteins were expressed using the RTS-100 expression system for 6 h at 25°C. Total and soluble fractions of the reaction mixture were separated by SDS-PAGE (left panel) and analyzed by immunoblotting (right panel) using rabbit antipeptide specific to 17 aa at the C terminus of botulinum type A heavy chain. Lane 1, negative control (no DNA); lane 2, total HcA-His₆; lane 3, soluble HcA-His₆; lane 4, total His₆-HcA; lane 5, soluble His₆-HcA; lane M, molecular mass marker.

FIG. 2.

Optimization of HcA expression in a cell-free system. Plasmid (0.5 mg) encoding HcA was used as a template in RTS-100 cell-free reaction at 30°C for 5 h with constant shaking (220 rpm). Expression was conducted in the absence (control) or presence of different additives as indicated, including the HSP-60 and HSP-70 chaperone system, protein disulfide isomerase (PDI), and oxidized (GSSG) and reduced (GSH) glutathione. Total protein (T) and soluble fraction (S) were separated by SDS-PAGE and analyzed by immunoblotting using rabbit antipeptide aa 1279 to 1295 of botulinum A.

FIG. 3.

Characterization of in vitro-expressed Hc proteins. (a) Purified preparations of HcA, HcB, and HcE were analyzed by SDS-PAGE. Lane 1, molecular mass marker (M); lane 2, HcA-His₆; lane 3, His₆-HcB; lane 4, HcE-His₆. (b) Purified HcA (lane 2) and botulinum A toxin complex (lane 3) were analyzed by immunoblotting using rabbit antipeptide aa 1279 to 1295 of botulinum A (left panel) and rabbit antitoxin A complex (right panel). The positions of HcA (lower arrow) and the botulinum A heavy chain (upper arrow) are marked.

FIG. 4.

Antitoxin antibody titer development in mice immunized with monovalent HcA, HcB, or HcE vaccine. Mice were immunized with three consecutive injections of alum-adsorbed HcA, HcB, or HcE vaccine. Ten days after the second and the third immunizations (Immun.), mice were bled and anti-homolog toxin titers were measured by ELISA. The geometric mean and geometric standard deviation are presented for each group.

FIG. 5.

Antitoxin antibody titer in mice immunized with monovalent and trivalent Hc vaccines. Mice were immunized with three consecutive injections containing 5 μg HcA, HcB, or HcE in alum (monovalent vaccine) or with a combined preparation containing 5 μg each HcA, HcB, and HcE (trivalent vaccine). Ten days after the third immunization, mice were bled and anti-toxin A, B, or E titers were measured by ELISA. The geometric mean and geometric standard deviation are presented for each group.