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Infection and Immunity, September 2001, p. 5709-5715, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5709-5715.2001

Candidate Vaccine against Botulinum Neurotoxin Serotype A Derived from a Venezuelan Equine Encephalitis Virus Vector System

John S. Lee,1,* Peter Pushko,1 Michael D. Parker,1 Mark T. Dertzbaugh,2 Leonard A. Smith,2 and Jonathan F. Smith1

Virology Division1 and Toxinology Division,2 U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011

Received 8 January 2001/Returned for modification 23 March 2001/Accepted 11 May 2001

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (HC) of the BoNT/A heavy chain was cloned into the replicon vector (HC-replicon). Cotransfection of BHK cells in vitro with the HC-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, HC VEE replicon particles (HC-VRP). Cells infected with HC-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of HC-VRP, mice were vaccinated with various doses of HC-VRP at different intervals. Mice inoculated subcutaneously with HC-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

* Corresponding author. Mailing address: Virology Division, USAMRIID, Fort Detrick, Frederick, MD 21702. Phone: (301) 619-4912. Fax: (301) 619-2290. E-mail: John.Lee{at}det.amedd.army.mil.

Infection and Immunity, September 2001, p. 5709-5715, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5709-5715.2001


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