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Special Issue "Botulinum Neurotoxins Antibody and Vaccine"

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Bacterial Toxins".

Deadline for manuscript submissions: closed (31 August 2017)

Special Issue Editors

Guest Editor
Prof. Dr. Jianlong Lou

Departments of Anesthesia and Pharmaceutical Chemistry, UCSF, San Francisco, CA, USA
Website | E-Mail
Interests: botulinum toxins; antibodies; monoclonal; sterigmatocystin antibodies
Guest Editor
Prof. Dr. James D. Marks

Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA
Website | E-Mail
Interests: Botulinum nerutoxin; intoxication therapy; therapeutic antibodies; immunotherapy

Special Issue Information

Dear Colleagues,

Botulinum neurotoxins (BoNTs) are true miracle protein molecules created by nature, which embody the biological functional beauty of yin and yang in the same structure. On the one hand, they are the most toxic substances known to human being, and are listed as one of the six highest-risk threat agents for bioterrorism in the USA, and they could cause botulism, a deadly human and animal disease if not treated timely. On the other hand, they are the key components of several widely used government approved medicines, such as Botox® and Myobloc®, for many clinical and cosmetic applications, with multi-billion dollar sales annually all over the world. To mitigate the potential risk of BoNTs, while making full use of their therapeutic powers for the ever-expanding list of human diseases, rationally-designed and applied antitoxins or vaccines seem to be the best option to accomplish this task. In this Special Issue of “Botulinum Neurotoxins Antibody and Vaccine”, the most up-to-date research and investigation on counter-measures for botulism are compiled, and the possible unwanted neutralizing antibody reaction issues in BoNT therapeutic targets are also discussed. Any format of antitoxin or vaccine for BoNT can be presented here, and a few reviews for the BoNT antibody or vaccine production will be selected as well.

Prof. Dr. Jianlong Lou
Prof. Dr. James D. Marks
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Toxins is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1500 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Botulinum neurotoxins (BoNTs)
  • Antibody
  • Vaccine
  • Bio-therat reduction
  • Monoclonal
  • Oligoclonal
  • Polyclonal
  • Antibody Engineering
  • Yeast display
  • Phage display
  • Hybridoma
  • Cell Culture
  • Molecular evolution
  • Epitope mapping
  • Immunoassay
  • Immunotherapy
  • Purification
  • Efficacy
  • Neutralization
  • Protection
  • Clearance
  • Pharmacokinetic
  • Biodistribution
  • Serotype
  • Subtype
  • Therapeutics
  • Mechanism
  • affinity
  • Animal model
  • Horse
  • Mouse Recombinant
  • Fragment
  • Epitope
  • Endopeptidase
  • protinomics
  • bio-sensing

Published Papers (10 papers)

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Research

Jump to: Review

Open AccessArticle Humoral Response of Buffaloes to a Recombinant Vaccine against Botulism Serotypes C and D
Toxins 2017, 9(10), 297; doi:10.3390/toxins9100297
Received: 31 August 2017 / Revised: 15 September 2017 / Accepted: 16 September 2017 / Published: 22 September 2017
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Abstract
Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the
[...] Read more.
Botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs), which are mainly produced by Clostridium botulinum and characterized by flaccid paralysis. The BoNTs C and D are the main serotypes responsible for botulism in animals, including buffaloes. Botulism is one of the leading causes of death in adult ruminants in Brazil due to the high mortality rates, even though botulism in buffaloes is poorly reported and does not reflect the real economic impact of this disease in Brazilian herds. Vaccination is reported as the most important prophylactic measure for botulism control, although there are no specific vaccines commercially available for buffaloes in Brazil. This study aimed to evaluate the humoral immune response of buffalo groups vaccinated with three different concentrations of recombinant proteins (100, 200, and 400 µg) against BoNTs serotypes C and D as well as to compare the groups to each other and with a group vaccinated with a bivalent commercial toxoid. The recombinant vaccine with a concentration of 400 μg of proteins induced the highest titers among the tested vaccines and was proven to be the best choice among the formulations evaluated and should be considered as a potential vaccine against botulism in buffalo. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism
Toxins 2017, 9(9), 269; doi:10.3390/toxins9090269
Received: 19 July 2017 / Revised: 29 August 2017 / Accepted: 30 August 2017 / Published: 3 September 2017
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Abstract
There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering
[...] Read more.
There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT/B1 HP, ciBoNT/C1 HP, ciBoNT/E1 HP and ciBoNT/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT/A Hc, BoNT/B Hc, BoNT/C Hc, BoNT/E Hc and BoNT/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT/Hc antigens resulted in increased survival rates at the toxin challenge levels used. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Efficacy and Safety of Letibotulinum Toxin A for the Treatment of Dynamic Equinus Foot Deformity in Children with Cerebral Palsy: A Randomized Controlled Trial
Toxins 2017, 9(8), 252; doi:10.3390/toxins9080252
Received: 12 July 2017 / Revised: 14 August 2017 / Accepted: 16 August 2017 / Published: 18 August 2017
Cited by 1 | PDF Full-text (1732 KB) | HTML Full-text | XML Full-text
Abstract
The objective of this clinical trial was to compare the efficacy and safety of letibotulinum toxin A and onabotulinum toxin A for improving dynamic equinus foot deformity in children with cerebral palsy (CP). In total, 144 children with spastic CP who had dynamic
[...] Read more.
The objective of this clinical trial was to compare the efficacy and safety of letibotulinum toxin A and onabotulinum toxin A for improving dynamic equinus foot deformity in children with cerebral palsy (CP). In total, 144 children with spastic CP who had dynamic equinus foot deformity were assigned randomly to the Botulax group (injection of letibotulinum toxin A) or the Botox group (injection of onabotulinum toxin A). The Physician’s Rating Scale (PRS), ankle plantar flexor spasticity using the Modified Tardieu Scale, the Gross Motor Function Measure (GMFM)-88, and the GMFM-66 were completed before injection and at 6, 12, and 24 weeks after injection. The PRS responder rate was 60.27% in the Botulax group and 61.43% in the Botox group at 12 weeks after treatment, and the lower limit of the 95% confidence interval for the between-group difference in responder rates was −17.16%, higher than the non-inferiority margin of −24.00%. The clinical efficacy and the safety profiles of the groups did not significantly differ. The results suggest that injection of letibotulinum toxin A is as effective and safe as that of onabotulinum toxin A for the treatment of dynamic equinus foot deformity in children with spastic CP. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E
Toxins 2017, 9(7), 230; doi:10.3390/toxins9070230
Received: 11 May 2017 / Accepted: 18 July 2017 / Published: 20 July 2017
Cited by 2 | PDF Full-text (1488 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization
[...] Read more.
Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations
Toxins 2017, 9(6), 180; doi:10.3390/toxins9060180
Received: 25 April 2017 / Revised: 24 May 2017 / Accepted: 27 May 2017 / Published: 29 May 2017
Cited by 1 | PDF Full-text (1022 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the
[...] Read more.
The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle RBC Adherence of Immune Complexes Containing Botulinum Toxin Improves Neutralization and Macrophage Uptake
Toxins 2017, 9(5), 173; doi:10.3390/toxins9050173
Received: 5 April 2017 / Revised: 12 May 2017 / Accepted: 15 May 2017 / Published: 19 May 2017
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Abstract
In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that
[...] Read more.
In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that the BoNT-neutralizing potency of mAbs can be improved through red blood cell (RBC) immunoadherence. For example, a fusion protein (FP) that adheres biotinylated mAbs to the RBC surface enabled a pair of mAbs to neutralize 5000 LD50 BoNT/A in the mouse protection assay. Here, we added two mAbs to that combination, creating a 4-mAb:FP complex that neutralized 40,000 LD50 BoNT/A in vivo, and analyzed functional correlates of neutralization. The FP enhanced potency of BoNT/A immune complexes, providing the greatest magnitude of benefit to the 4-mAb combination. RBC binding of a BoNT/A complexed with 4-mAb:FP exhibited a bi-phasic clearance process in vivo. Most of the complexes were cleared within five minutes; the rest were cleared gradually over many hours. Peritoneal macrophages showed better uptake of the 4-mAb complex than the 3-mAb complex, and this was not affected by the presence of the FP. However, the addition of RBCs to the 4-mAb:FP BoNT/A doubled macrophage uptake of the complexes. Lastly, the 4-mAb:FP BoNT/A complex synergistically induced M2 macrophage polarization, as indicated by IL-10 expression, whether or not RBCs were present. RBC-targeted immunoadherence through the FP is a potent enhancer of mAb-mediated BoNT/A neutralization in vivo, and can have positive effects on BoNT/A sequestration, immune complex uptake, and macrophage activation. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins
Toxins 2016, 8(10), 276; doi:10.3390/toxins8100276
Received: 8 August 2016 / Revised: 14 September 2016 / Accepted: 19 September 2016 / Published: 24 September 2016
Cited by 5 | PDF Full-text (2413 KB) | HTML Full-text | XML Full-text
Abstract
Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming,
[...] Read more.
Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessArticle Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody
Toxins 2016, 8(9), 257; doi:10.3390/toxins8090257
Received: 5 July 2016 / Revised: 15 August 2016 / Accepted: 22 August 2016 / Published: 12 September 2016
Cited by 3 | PDF Full-text (1237 KB) | HTML Full-text | XML Full-text
Abstract
Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and
[...] Read more.
Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Review

Jump to: Research

Open AccessReview The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies
Toxins 2017, 9(10), 309; doi:10.3390/toxins9100309
Received: 11 August 2017 / Revised: 15 September 2017 / Accepted: 16 September 2017 / Published: 2 October 2017
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Abstract
The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and
[...] Read more.
The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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Open AccessReview Vaccines against Botulism
Toxins 2017, 9(9), 268; doi:10.3390/toxins9090268
Received: 8 August 2017 / Revised: 30 August 2017 / Accepted: 30 August 2017 / Published: 2 September 2017
PDF Full-text (1177 KB) | HTML Full-text | XML Full-text
Abstract
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
[...] Read more.
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. Full article
(This article belongs to the Special Issue Botulinum Neurotoxins Antibody and Vaccine)
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