Oral vaccination with an adenovirus-vectored vaccine protects against botulism.
ABSTRACT: We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1×10(4) to 1×10(7)plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2×10(7)pfu adenovirus, have shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2×10(6)pfu adenovirus or greater were completely protected against challenge with 100×MLD(50) of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism.
Project description:Botulinum neurotoxins cause botulism, a neuroparalytic disease in humans and animals. We constructed a replication-incompetent adenovirus encoding a synthesized codon-optimized gene for expression of the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). This recombinant human serotype 5 adenoviral vector (Ad5) was evaluated as a genetic vaccine candidate against botulism caused by BoNT/C in a mouse model. A one-time intramuscular injection with 10(5) to 2 x 10(7)pfu of adenoviral vectors elicited robust serum antibody responses against H(C)50 of BoNT/C as assessed by ELISA. Immune sera showed high potency in neutralizing the active BoNT/C in vitro. After a single dose of 2 x 10(7)pfu adenoviral vectors, the animals were completely protected against intraperitoneal challenge with 100 x MLD(50) of active BoNT/C. The protective immunity appeared to be vaccine dose-dependent. The anti-toxin protective immunity could last for at least 7 months without a booster injection. In addition, we observed that pre-existing immunity to the wild-type Ad5 in the host had no significant influence on the protective efficacy of vaccination. The data suggest that an adenovirus-vectored genetic vaccine is a highly efficient prophylaxis candidate against botulism.
Project description:A replication-incompetent adenoviral vector encoding the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C) was evaluated as a mucosal vaccine against botulism in a mouse model. Single intranasal inoculation of the adenoviral vector elicited a high level of H(C)50-specific IgG, IgG1 and IgG2a in sera and IgA in mucosal secretions as early as 2 weeks after vaccination. The antigen-specific serum antibodies were maintained at a high level at least until the 27th week. Immune sera showed high potency in neutralizing BoNT/C as indicated by in vitro toxin neutralization assay. The mice receiving single dose of 2 x 10(7) p.f.u. (plaque-forming unit) of adenoviral vector were completely protected against challenge with up to 10(4) x MLD(50) of BoNT/C. The protective immunity showed vaccine dose dependence from 10(5) to 2 x 10(7) p.f.u. of adenoviral vector. In addition, animals receiving single intranasal dose of 2 x 10(7) p.f.u. adenoviral vector could be protected against 100 x MLD(50) 27 weeks after vaccination. Animals with preexisting immunity to adenovirus could also be vaccinated intranasally and protected against lethal challenge with BoNT/C. These results suggest that the adenoviral vector is a highly effective gene-based mucosal vaccine against botulism.
Project description:Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin.
Project description:Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HCN) and a receptor binding domain (HCC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHCN/A1 and HCC/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >106-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1W. M-BoNT/A1W vaccination protected against challenge by 106 LD50 Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1W surviving BoNT challenge had dominant antibody responses to the LCHCN domain, but varied antibody responses to HCC. Sera from mice vaccinated with M-BoNT/A1W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.
Project description:The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR). Recombinant HCR subunits have been used to develop the next generation of BoNT vaccines. Using structural studies and the known entry properties of BoNT/A, an HCR subunit vaccine against BoNT/A that contained the point mutation W1266A within the ganglioside binding pocket was designed. HCR/A(W1266A) did not enter primary neurons, and the crystal structure of HCR/A(W1266A) was virtually identical to that of wild-type HCR/A. Using a mouse model, experiments were performed using a high-dose vaccine and a low-dose vaccine. At a high vaccine dose, HCR/A and HCR/A(W1266A) elicited a protective immune response to BoNT/A challenge. At the low-dose vaccination, HCR/A(W1266A) was a more protective vaccine than HCR/A. ?-HCR IgG titers correlated with protection from BoNT challenge, although titers to block HCR/A entry were greater in serum in HCR/A-vaccinated mice than in HCR/A(W1266A)-vaccinated mice. This study shows that removal of receptor binding capacity enhances potency of the subunit HCR vaccine. Vaccines that lack receptor binding capacity have the added property of limited off-target toxicity.
Project description:Influenza virus is a negative segmented RNA virus without DNA intermediate. This makes it safer as a vaccine delivery vector than most DNA viruses that have potential to integrate their genetic elements into host genomes. In this study, we developed a universal influenza viral vector, expressing the receptor binding subdomain of botulinum neurotoxin A (BoNT/A). We tested the growth characters of the engineered influenza virus in chicken eggs and Madin-Darby canine kidney epithelial cells (MDCK), and showed that it can be produced to a titer of 5?×?10(6) plaque forming unites/ml in chicken eggs and MDCK cells. Subsequently, mice intranasally vaccinated with the engineered influenza virus conferred protection against challenge with lethal doses of active BoNT/A toxin and influenza virus. Our results demonstrated the feasibility to develop a dual purpose nasal vaccine against both botulism and influenza.
Project description:Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD<sub>50</sub> of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (<i>Cavia porcellus</i>) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD<sub>50</sub> of BoNT/A1 and 116 LD<sub>50</sub> of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.
Project description:Botulinum neurotoxins (BoNTs) are potent neuroparalytic toxins that cause mortality through respiratory paralysis. The approved medical countermeasure for BoNT poisoning is infusion of antitoxin immunoglobulins. However, antitoxins have poor therapeutic efficacy in symptomatic patients; thus, there is an urgent need for treatments that reduce the need for artificial ventilation. We report that the US Food and Drug Administration-approved potassium channel blocker 3,4-diaminopyridine (3,4-DAP) reverses respiratory depression and neuromuscular weakness in murine models of acute and chronic botulism. In ex vivo studies, 3,4-DAP restored end-plate potentials and twitch contractions of diaphragms isolated from mice at terminal stages of BoNT serotype A (BoNT/A) botulism. In vivo, human-equivalent doses of 3,4-DAP reversed signs of severe respiratory depression and restored mobility in BoNT/A-intoxicated mice at terminal stages of respiratory collapse. Multiple-dosing administration of 3,4-DAP improved respiration and extended survival at up to 5 LD50 BoNT/A. Finally, 3,4-DAP reduced gastrocnemius muscle paralysis and reversed respiratory depression in sublethal models of serotype A-, B-, and E-induced botulism. These findings make a compelling argument for repurposing 3,4-DAP to symptomatically treat symptoms of muscle paralysis caused by botulism, independent of serotype. Furthermore, they suggest that 3,4-DAP is effective for a range of botulism symptoms at clinically relevant time points.
Project description:A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission's Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.
Project description:A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission’s Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.