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

Abstract

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 (H C ) of the BoNT/A heavy chain was cloned into the replicon vector (H C -replicon). Cotransfection of BHK cells in vitro with the H C -replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagationdeficient, H C VEE replicon particles (H C -VRP). Cells infected with H C -VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H C -VRP, mice were vaccinated with various doses of H C -VRP at different intervals. Mice inoculated subcutaneously with H C -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.Botulism is a disease resulting from the activity of botulinum neurotoxins (BoNT) produced by Clostridium botulinum on the transmission of neuromuscular stimuli (8,22,23). The blockage of stimuli produces neuromuscular weakness and flaccid paralysis, which can lead to respiratory failure and death. Food poisoning, infant botulism, and wound botulism are the three most common BoNT diseases affecting humans. The BoNT consists of two polypeptides bound by a disulfide bond, a heavy chain of about 100 kDa and a light chain of about 50 kDa. Seven different serotypes (A through G) of BoNT have been characterized. Previous research has shown that polyclonal antibodies to one serotype can block the effects of the homologous serotype but not of heterologous serotypes (20). The current human vaccine, which is administered under Investigational New Drug status to at-risk laboratory personnel, contains five of the seven serotypes (A to E) and is formulated as a toxoid. The toxoid vaccine is given as a primary series of three inoculations given at 0, 2, and 12 weeks, followed by a booster at 1 year. Since the vaccine is reactogenic in up to 20% of the recipients and contains only five of the seven serotypes, an improved vaccine would be preferable.Venezuelan equine encephalitis (VEE) virus is a member of the Alphavirus genus in the Togaviridae family. Alphaviruses contain a 42S single-stranded positive-sense RNA genome encoding four nonstructural proteins (providing the replicase and transcriptase function) and three structural proteins (capsid, E1, and E2). The nonstructural proteins are tran...

Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine

“…These constructs not only yielded high levels of H C fragments, as judged by immunofluorescence and immunoblotting analysis, but also protected mice against BoNT/A or BoNT/C challenge after 2 or 3 injections with 10 7 infectious units (i.u.) of VEE [39][40][41]. However, a single dose of genetic vaccine was not fully protective against botulism in these studies.…”

Viral vectors for use in the development of biodefense vaccines

“…The antibody responses and protection were not diminished following sequential vaccination and no anti-VEEV antibody responses were detected in the BALB/c mice used in the experiment. In contrast, Swiss mice inoculated with a different VEEV-vectored vaccine did produce neutralizing antibodies against VEEV but the neutralizing antibodies did not interfere with subsequent booster vaccinations [92]. Sequential inoculation of animals with SINV-replicons expressing SEOV-S then with SINV-replicons expressing the lacZ gene elicited antibodies to each protein, although antibody levels were lower in the mice with preexisting anti-SINV antibodies [73].…”

“…A candidate vaccine against BoNT serotype A (BoNT/A) was developed by cloning the non-toxic 50-kDa carboxy-terminal fragment (Hc) from the heavy chain of BoNT/A (BoNT/A Hc) into the VEEV-replicon vector [92]. Vaccinated mice were protected from an i.p.…”

Vaccines against botulism

“…VEE has been examined as a vaccine vector for BoNT, anthrax, and Marburg virus [33,34]. Lee et al inserted the gene encoding HC/A downstream of the 26S promotor in the VEE genome, replacing genes which encode for viral structural proteins and rendering the virion replicon particles (HC/A-VRP) replication defective [33]. A HC/A-VRP vaccination regimen with two doses of 10 7 Units protected mice against challenge by 10,000 MLD 50 Units of BoNT/A [33] with protection extending for up to one-year post vaccination.…”

“…Lee et al inserted the gene encoding HC/A downstream of the 26S promotor in the VEE genome, replacing genes which encode for viral structural proteins and rendering the virion replicon particles (HC/A-VRP) replication defective [33]. A HC/A-VRP vaccination regimen with two doses of 10 7 Units protected mice against challenge by 10,000 MLD 50 Units of BoNT/A [33] with protection extending for up to one-year post vaccination. Mice vaccinated with HC/A-VRP and replicon particles containing genes for Bacillus anthracis mature protective antigen (PA-VRP) and Marburg virus glycoprotein (MBGV-GP-VRP) survived challenge by 1000 MLD 50 Units of BoNT/A several months post vaccination [34].…”