RESUMEN
INTRODUCTION: Producing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. OBJECTIVE: Considering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. METHODS: Subcutaneously, 18 Texel sheep received two doses (200 µg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). RESULTS: Both formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. CONCLUSION: While adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Neutralizantes , Vacunas Bacterianas , Botulismo , Enterotoxemia , Animales , Botulismo/prevención & control , Botulismo/veterinaria , Botulismo/inmunología , Ovinos , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Anticuerpos Antibacterianos/sangre , Enterotoxemia/prevención & control , Enterotoxemia/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Enfermedades de las Ovejas/prevención & control , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Inmunoglobulina G/sangre , Escherichia coli/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , FemeninoRESUMEN
Botulism is one of the most serious food intoxications, manifesting as prolonged paralytic conditions. This disease is usually the result of the consumption of poor quality canned or smoked foods, so the inhabitants of many countries of the world are exposed to the risk of this kind of poisoning every year. In view of the severity of poisonings caused by botulinum neurotoxins, monoclonal antibodies (mAbs) show great promise because of their targeting action, lack of allergic reactions and serum sickness. The use of a cocktail of mAbs increases the "functional specificity" of their mixture, allowing them to bind to the active domains of different toxin chains and block their action. In this work, we obtained 14 murine mAbs to the catalytic and receptor-binding domain of botulinum toxin type A. The Sp2/0-Ag14 murine myeloma cell line and splenocytes from immunized mice of the BALB/c line were used as fusion partners. We have shown that the selected cocktail of three antibodies neutralizes native toxin more effectively than antibodies separately-complete neutralization is achieved at a toxin dose of 3LD50 and partial neutralization at 5LD50. We presume that this cocktail may be promising as a prototype for the creation of a therapeutic drug capable of neutralizing the toxin in the blood of patients.
Asunto(s)
Anticuerpos Monoclonales , Toxinas Botulínicas Tipo A , Ratones Endogámicos BALB C , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/inmunología , Toxinas Botulínicas Tipo A/inmunología , Toxinas Botulínicas Tipo A/toxicidad , Botulismo/tratamiento farmacológico , Botulismo/inmunología , Ratones , Línea Celular Tumoral , Femenino , Anticuerpos Neutralizantes/inmunologíaRESUMEN
Among all natural and synthetic toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botulinum in an anaerobic environment, are the most toxic polymer proteins. Currently, the most effective modalities for botulism prevention and treatment are vaccination and antitoxin use, respectively. However, these modalities are associated with long response time for active immunization, side effects, and donor limitations. As such, the development of more promising botulism prevention and treatment modalities is warranted. Here, we designed an mRNA encoding B9-hFc - a heavy-chain antibody fused to VHH and human Fc that can neutralize BoNT serotype B (BoNT/B) effectively - and assessed its expression in vitro and in vivo. The results confirmed that our mRNA demonstrates good expression in vitro and in vivo. Moreover, a single mRNA lipid nanoparticle injection effectively prevents BoNT/B intoxication in vivo, with effects comparable to those of protein antibodies. In conclusion, we explored and clarified whether mRNA drugs encoding neutralizing antibodies prevent BoNT/B intoxication. Our results provide an efficient strategy for further research on the prevention and treatment of intoxication by botulinum toxin.
Asunto(s)
Anticuerpos Neutralizantes , Toxinas Botulínicas Tipo A , Botulismo , ARN Mensajero , Anticuerpos Neutralizantes/inmunología , Animales , Botulismo/prevención & control , Botulismo/inmunología , Toxinas Botulínicas Tipo A/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Ratones , Humanos , Femenino , Nanopartículas , Ratones Endogámicos BALB C , Anticuerpos Antibacterianos/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , LiposomasRESUMEN
Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.
Asunto(s)
Anticuerpos Neutralizantes , Toxinas Botulínicas Tipo A , Botulismo , Animales , Femenino , Humanos , Ratones , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Toxinas Botulínicas Tipo A/inmunología , Botulismo/inmunología , Botulismo/terapia , Cadenas Pesadas de Inmunoglobulina/inmunología , Biblioteca de Péptidos , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most toxic known protein and the causative agent of human botulism. BoNTs have similar structures and functions, comprising three functional domains: catalytic domain (L), translocation domain (HN), and receptor-binding domain (Hc). In the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. The EL-HN fragment (L and HN domains of BoNT/E) retained the enzymatic activity without in vivo neurotoxicity. Extensive investigations showed EL-HN functional fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Further experiments demonstrated the EL-HN provided a superior protective effect compared with the EHc or EHc and EL-HN combination. Thus, the EL-HN played an important role in immune protection against BoNT/E and could provide an excellent platform for the design of botulinum vaccines and neutralizing antibodies. The EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Botulismo/inmunología , Botulismo/prevención & control , Clostridium botulinum/inmunología , Neurotoxinas/toxicidad , Animales , Clostridium botulinum/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Sustancias Protectoras/administración & dosificación , SerogrupoRESUMEN
Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Toxinas Botulínicas/inmunología , Animales , Botulismo/inmunología , Femenino , Humanos , Ratones , SerogrupoRESUMEN
OBJECTIVES: To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. RESULTS: In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. CONCLUSIONS: Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.
Asunto(s)
Proteínas Bacterianas/inmunología , Clostridium botulinum tipo B/inmunología , Animales , Proteínas Bacterianas/metabolismo , Botulismo/diagnóstico , Botulismo/inmunología , Botulismo/prevención & control , Clostridium botulinum/clasificación , Clostridium botulinum/inmunología , Clostridium botulinum tipo B/aislamiento & purificación , Clostridium botulinum tipo B/metabolismo , Reacciones Cruzadas , Medios de Cultivo/metabolismo , Sueros Inmunes/inmunología , Ratones , ProteómicaRESUMEN
As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Toxinas Botulínicas Tipo A/inmunología , Botulismo/prevención & control , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Botulismo/inmunología , Botulismo/microbiología , Línea Celular , Clonación Molecular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Mapeo Epitopo , Femenino , Humanos , Hibridomas , Inmunización , Masculino , Ratones Endogámicos BALB C , Pruebas de Neutralización , Factores de TiempoRESUMEN
Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Toxinas Botulínicas/antagonistas & inhibidores , Botulismo/prevención & control , Camélidos del Nuevo Mundo/inmunología , Neuronas/efectos de los fármacos , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología , Anticuerpos de Dominio Único/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Toxinas Botulínicas/administración & dosificación , Toxinas Botulínicas/inmunología , Botulismo/inmunología , Botulismo/microbiología , Células Cultivadas , Modelos Animales de Enfermedad , Inmunización , Masculino , Ratones , Neuronas/metabolismo , Neuronas/patología , Péptido Hidrolasas/administración & dosificación , Péptido Hidrolasas/inmunología , Inhibidores de Proteasas/inmunología , Ratas , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Botulismo/prevención & control , Anticuerpos ampliamente neutralizantes/farmacología , Clostridium botulinum/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Toxinas Botulínicas Tipo A/inmunología , Botulismo/inmunología , Botulismo/microbiología , Anticuerpos ampliamente neutralizantes/inmunología , Clostridium botulinum/inmunología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Epítopos , Femenino , Humanos , Hibridomas , Ratones , Pruebas de Neutralización , Unión ProteicaRESUMEN
Botulism is a neuroparalytic intoxication, usually fatal, caused by the botulinum toxins (BoNTs). Vaccination is the best-known strategy to prevent this disease in ruminants. Serotypes C and D and their variants CD and DC are the main types responsible for botulism in bovine and buffaloes in Brazil and cattle in Japan and Europe. Brazil has a herd of approximately 1.39 million buffaloes and is the largest producer in the Western world. This study aimed to assess the humoral immune response of buffaloes during the 12-month period after vaccination against BoNT serotypes C and D with a recombinant vaccine in three different concentrations (100, 200, and 400⯵g) of non-purified recombinant proteins (Vrec) and also with a bivalent commercial toxoid (Vcom). Vrec400 was the best vaccine among those tested because it induced higher levels of antibodies and maintained higher levels of antibodies for the longest time, while Vrec200 could be considered the most cost-effective vaccine for large-scale production. None of the vaccines were able to promote continuous immunological protection within the timeframe proposed by the current Brazilian vaccination protocol. Further studies should focus on vaccine adjustments to ensure continued humoral protection against botulism.
Asunto(s)
Botulismo/terapia , Búfalos/microbiología , Inmunidad Humoral , Vacunación/veterinaria , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos , Anticuerpos Neutralizantes , Vacunas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Botulismo/inmunología , Botulismo/veterinaria , Búfalos/inmunología , Bovinos , Clostridium/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.
Asunto(s)
Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Antitoxina Botulínica/química , Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Toxinas Botulínicas/antagonistas & inhibidores , Botulismo/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antitoxina Botulínica/inmunología , Toxinas Botulínicas/química , Toxinas Botulínicas/inmunología , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/inmunología , Botulismo/tratamiento farmacológico , Calor , Humanos , Estabilidad ProteicaRESUMEN
Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.
Asunto(s)
Toxinas Botulínicas/genética , Clonación Molecular/métodos , Clostridium botulinum/genética , Animales , Anticuerpos Neutralizantes/inmunología , Toxinas Botulínicas/química , Toxinas Botulínicas/inmunología , Botulismo/inmunología , Botulismo/microbiología , Clostridium botulinum/química , Clostridium botulinum/inmunología , Escherichia coli/genética , Ratones , Ratones Endogámicos BALB C , ConejosRESUMEN
OBJECTIVES: We undertook an open-label, uncontrolled study of investigational recombinant botulinum vaccine for botulinum neurotoxin (BoNT) serotypes A and B (rBV A/B) to assess its safety and immunogenicity in healthy volunteers who had been previously immunized with investigational pentavalent botulinum toxoid. Study participants who wished to do so could donate their hyperimmune plasma for production of Human Botulism Immune Globulin Intravenous (BIG-IV, BabyBIG®). STUDY DESIGN: A single 0.5â¯ml (mL), 40-microgram intramuscular injection of rBV A/B was administered to study participants. Post-vaccination sera collected at approximately 2-week intervals were evaluated for anti-BoNT/A and anti-BoNT/B neutralizing antibody concentrations (NAC). Local and systemic treatment-emergent adverse events (TEAEs) were identified by clinical and laboratory monitoring for 12â¯weeks post-vaccination with a final telephone follow-up for additional safety assessment at 6â¯months. The primary endpoint for immunogenicity was a ≥4-fold rise in NAC in ≥50% of participants by Week 4 post-vaccination. RESULTS: All 45 enrolled participants completed the study. Forty-two of 45 participants (93.3%) experienced at least one TEAE. Overall, 138 of 218 (63.3%) reported TEAEs were treatment-related, the majority of which were mild injection-site reactions. No serious or unexpected adverse events occurred. The study achieved its primary immunogenicity endpoint with 37/45 (82.2%) participants and 39/45 (86.7%) participants having a ≥4-fold rise in NAC to anti-BoNT/A and to anti-BoNT/B, respectively, by Week 4 post-vaccination. CONCLUSION: A single 0.5â¯mL dose of rBV A/B was safe, well-tolerated and immunogenic in participants previously immunized with pentavalent botulinum toxoid. The tolerability and immunogenicity characteristics of rBV A/B vaccination of individuals with existing BoNT immunity support its potential future use to provide occupational protection to botulism laboratory workers. Almost all study participants donated hyperimmune plasma for production of BIG-IV. ClinicalTrials.gov registration number: NCT01701999.
Asunto(s)
Vacunas Bacterianas/inmunología , Botulismo/inmunología , Botulismo/prevención & control , Clostridium botulinum/inmunología , Inmunogenicidad Vacunal , Vacunas Sintéticas/inmunología , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/efectos adversos , Toxinas Botulínicas/inmunología , Agentes Comunitarios de Salud , Femenino , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversosRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Botulismo/inmunología , Botulismo/prevención & control , Clostridium botulinum/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Inmunización , Ratones , Neuronas/inmunología , Neuronas/metabolismo , Proteínas RecombinantesRESUMEN
Diseases triggered by microorganisms can be controlled by vaccines, which need neutralizing antigens. Hence, it is very crucial to identify extremely efficient immunogens for immune prevention. Botulism, a fatal neuroparalytic disease, is caused by botulinum neurotoxins produced by the anaerobic, Gram-positive spore-forming bacteria, Clostridium botulinum. Food-borne botulism and iatrogenic botulism are caused by botulinum toxin. Wound botulism, infant botulism, and adult intestinal botulism are caused by primarily C. botulinum followed by secondary intoxication. To identify protective antigens, whole cell proteome of C. botulinum type B was separated by two-dimensional gel electrophoresis. 2-D gel of whole cell proteins was probed with hyper immune sera of whole cell proteins of C. botulinum types A, E, and F. Six cross immunoreactive proteins were identified. These immunoreactive proteins will be further tested for developing vaccines and serodiagnostic markers against botulism.
Asunto(s)
Toxinas Botulínicas/química , Botulismo/microbiología , Clostridium botulinum/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Botulismo/inmunología , Clostridium botulinum/genética , Clostridium botulinum/inmunología , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Because the mucosa is the major entry route for most pathogens, the development of mucosal vaccines is a rational approach for protecting against these undesired agents. Mucosal administration of vaccine antigen is useful for non-infectious chronic diseases as well, because of its advantages over injection routes, including comparable efficacy in the induction of systemic immune responses, less pain, and no risk of adverse events at the injection site. However, because it is difficult to effectively induce and regulate antigen-specific mucosal and systemic immune responses when antigen alone is mucosally administered, an appropriate form of mucosal delivery vehicle must be used. Antigen delivery systems involving nanogels, which act as artificial chaperones and mucosal adhesives, are a promising approach to overcoming this problem. Here, we introduce current perspectives regarding the development of nanogel-based nasal vaccines for both infectious and lifestyle-related diseases.
Asunto(s)
Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/terapia , Estilo de Vida , Mucosa Nasal/inmunología , Vacunas/administración & dosificación , Animales , Antígenos/administración & dosificación , Botulismo/inmunología , Botulismo/prevención & control , Enfermedad Crónica , Clostridium botulinum/inmunología , Sistemas de Liberación de Medicamentos , Geles , Humanos , Hipertensión/inmunología , Hipertensión/terapia , Nanoestructuras/administración & dosificación , Obesidad/inmunología , Obesidad/terapia , Vacunas Neumococicas/administración & dosificaciónRESUMEN
BACKGROUND: There are currently no licensed vaccines available for prevention of botulism in humans. The vaccination is not desirable due to expanding therapeutic indications of botulinum toxins. The only available specific treatment for botulism is antitoxin to remove circulating toxin, thus, preventing further neuronal damage. BAT® (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)) has been developed and its therapeutic efficacy evaluated against botulinum neurotoxin serotype A (BoNT/A) in Rhesus macaques. METHODS AND FINDINGS: In a post-exposure prophylaxis (PEP) study, animals were exposed to 4x LD50/kg of BoNT/A and administered intravenously with either BAT (1x or 0.1x scaled human dose), or placebo at 4 hours post-exposure. The animals were monitored for 14 days. For the therapeutic intervention studies, animals were exposed to a 1.7x LD50/kg of BoNT/A and treated intravenously with either placebo or BAT at a 1x scaled human dose at the onset of clinical signs. Animals were monitored on an hourly basis for 14 or 21 days. In the PEP study, all animals tolerated equine based antitoxin without any adverse clinical signs. A 100% survival was observed in groups treated with the BAT compared to 0% survival in those treated with the placebo (p<0.001, Fisher's exact test). BAT antitoxin prevented the development of signs of neurotoxicity of botulinum toxin. In a therapeutic study, treatment with the BAT at scaled 1x human dose after the onset of clinical signs significantly enhanced survival compared to the placebo (46.6% vs. 0%, p<0.0001, Fisher's exact test). Additionally, treatment with the BAT delayed the progression of signs (muscular weakness, respiratory distress, oral/nasal discharge) of toxin intoxication and reduced the severity of the disease. CONCLUSIONS: A single dose of BAT, when administered to symptomatic monkeys, resulted in a statistically significant survival benefit compared to the placebo. Additionally, BAT completely protected monkeys from the clinical signs of intoxication and subsequent death when administered as PEP treatment. These data in part supported the licensure of BAT under the Animal Rule in the United States by the Food and Drug Administration.
Asunto(s)
Antitoxina Botulínica/inmunología , Antitoxina Botulínica/uso terapéutico , Botulismo/inmunología , Botulismo/prevención & control , Enfermedades de los Monos/inmunología , Enfermedades de los Monos/prevención & control , Animales , Toxinas Botulínicas/toxicidad , Intervalos de Confianza , Caballos , Estimación de Kaplan-Meier , Macaca mulatta , Placebos , Profilaxis Posexposición , Índice de Severidad de la Enfermedad , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND AND AIMS: Botulinum toxin (Botox) injections are used as a cosmetic treatment to decrease wrinkles in face and chin. Being a neurotoxic agent it minimizes muscle activity, while side effects are usually rare. This article subsequently presents one case of these rare effects. CASE: A 30-year-old woman presenting with ptosis, diplopia, dysarthria, dysphagia and muscle weakness was admitted to our hospital. She had no history of disease. For cosmetic reasons, she had three Botox injections during the preceding months. On physical examination, muscle weakness 4/5 (cervical extensor, ocular and pharynx) was detected and a diagnosis of myasthenia gravis was made. Protective artificial ventilation was necessary. As a consequence, eight sessions of 2.5 L volume Therapeutic Plasma Exchange (TPE) were applied using normal saline/albumin as substitute. Due to TPE, her muscle force and clinical condition improved. Artificial ventilation could be stopped. CONCLUSIONS: Clinical symptoms of myasthenia gravis and systemic Botox effects are very similar. This should be taken into consideration during medical history taking. The injection of high doses of Botox (more than 200 units in every injection) or boostering within less than one month is dangerous. (Botox BCC2024). Systemic side effects can be treated using TPE to lower the circulating dose of Botox.
Asunto(s)
Inhibidores de la Liberación de Acetilcolina/efectos adversos , Toxinas Botulínicas Tipo A/efectos adversos , Botulismo/terapia , Técnicas Cosméticas/efectos adversos , Miastenia Gravis/terapia , Intercambio Plasmático , Inhibidores de la Liberación de Acetilcolina/administración & dosificación , Adulto , Autoanticuerpos/sangre , Biomarcadores/sangre , Toxinas Botulínicas Tipo A/administración & dosificación , Botulismo/sangre , Botulismo/inducido químicamente , Botulismo/inmunología , Femenino , Humanos , Inyecciones Subcutáneas , Miastenia Gravis/sangre , Miastenia Gravis/complicaciones , Miastenia Gravis/inmunología , Receptores Nicotínicos/inmunología , Resultado del TratamientoRESUMEN
As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.