DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Shyu, Rong-Hwa; Shaio, Men‐Fang; Tang, Shiao-Shek; Shyu, Huey-Fen; Lee, Chi‐Feng; Tsai, Meng-Hung; Smith, Jason; Huang, Hsin-Hsien; Wey, Jiunn-Jye; Huang, Jan-Ling; Chang, Hsin-Hou

doi:10.1007/bf02255918

Cited by 35 publications

(13 citation statements)

References 52 publications

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“…Strong immune responses have been obtained with transmembrane, secreted, or intracellular proteins. Several studies have indicated that a secreted form of the antigen leads to a stronger immune response and more neutralizing antibodies (2,12), although conflicting results have also been reported (43). Since the FcBoNT/A fragment is not naturally secreted, we fused the murine erythropoietin or human secreted alkaline phosphatase secretion signal 5Ј of the FcBoNT/A cDNA sequence.…”

Section: Discussionmentioning

confidence: 99%

Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer

et al. 2009

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Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.

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Section: Discussionmentioning

confidence: 99%

Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer

et al. 2009

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“…Another approach for developing BoNT vaccines has focused on "naked" DNA vaccine vectors. Two research groups have demonstrated that DNA-based vaccines can partially protect animals from challenge with BoNT/A (3,18).…”

Section: Discussionmentioning

confidence: 99%

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

Lee¹,

Pushko²,

Parker³

et al. 2001

Infect Immun

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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...

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“…Such results will provide important information for the development of safe and effective botulism vaccines. In addition to the currently used toxoids, some of the potential botulism vaccine candidates, such as recombinant vaccines (7) and DNA vaccines (32), have been developed. Using overlapping 19-residue peptides from residue 855 to 1296 of BTx-A, Atassi et al identified six synthetic peptides that could bind with human antitoxin antibodies and that may be useful as immunogens to induce immunity against BTx-A in the future (2).…”

Section: Vol 67 2001mentioning

confidence: 99%

Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on theClostridium botulinumNeurotoxin Type A

Yeh

Huang

et al. 2001

Appl Environ Microbiol

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Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10 3 and 10 4 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D 5 ) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K 5 ). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

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DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Cited by 35 publications

References 52 publications

Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer

Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer

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

Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on theClostridium botulinumNeurotoxin Type A

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