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Toxoids

Introduction

Toxoid is an inactivated toxin whose toxicity has been eliminated by chemical or heat treatment, while other properties, typically immunogenicity, are maintained. Toxins are secreted by bacteria, whereas toxoids are altered form of toxins. Thus, toxoids are among the most common of antivirulence vaccines. By immunizing against bacteria virulence factors, the body generates defensive measures against bacterial mechanisms of attack, decreasing their invasiveness. Currently, bacterial toxoids that are clinically used include vaccines against certain bacterial diseases. At the same time, Toxoids can be exploited as protein carriers to enhance the immunogenicity of small molecules. In addition, they can also be used to produce safer human antitoxins.

Classification

Toxoid proteins are biologically inactivated forms of native toxins. Toxoid vaccines are based on the toxin produced by certain bacteria, such as Clostridium tetani, Corynebacterium diphtheriae, Clostridium botulinum.

Organism Toxoid
Clostridium tetani Tetanus toxoid
Corynebacterium diphtheriae Diphtheria toxoid
Clostridium botulinum Botulinum toxoid
Clostridioides difficile C. difficile toxoid
Bordetella pertussis B. pertussis toxoid
Staphylococcus aureus S. aureus toxoid

Toxoid Vaccines

The invasion of toxins into the bloodstream is the main cause of disease symptoms. This technique is reserved for diseases in which the secreted toxins are the main cause of the illness. Protein-based toxins become harmless (toxoid) and are used as antigens in vaccines to elicit immunity. Vaccination with toxoids induces anti-toxoid antibodies that are able to bind with the toxin and neutralize its deleterious effects. Toxoid vaccines are relatively stable and safe. The vaccine antigens are less susceptible to changes in temperature, humidity and light, and they are not actively multiplying and do not spread to unimmunized individuals. Procedures for the production of toxoid vaccines ought to be strictly controlled to achieve detoxification/inactivation without excessive modification of the antigenic epitope structure.  Such “detoxified” toxins are harmless/safe and are used as vaccines. The most often used toxoid is tetanus toxoid (TT) and diphtheria toxoids (DT), but other proteins are also used occasionally.

The production steps in a typical clostridial vaccine production process

Fig. 1 The production steps in a typical clostridial vaccine production process (Nicolas E. Zaragoza, et al. 2019)

  • Tetanus toxoid

Two types of tetanus toxoid are available in the United States: fluid and adsorbed. The adsorbed vaccines contain less than 1.25 mg of aluminum and 4 to 10 flocculation units (Lf) of toxoid per 0.5-ml dose. The fluid preparations contain 4 to 5 Lf of toxoid. All tetanus toxoids in the United States contain 0.02 percent formaldehyde and 0.1 percent thimerosal. Currently, commercial tetanus toxoid is produced by culturing C. tetani in liquid medium and transforming the purified toxin with 40 percent formaldehyde at 37°C. In the United States, tetanus toxoid vaccines are available as a single tetanus toxoid vaccine (TT) and in combination with diphtheria toxoid as DT/Td, acellular pertussis as DTaP/Tdap, and as DTaP with other antigens such as Haemophilus influenzae B (HiB) conjugate.

  • Diphtheria toxoid

The first vaccine against diphtheria was developed in the early 1800s and was widely used in the United States as early as 1914. The vaccine consisted of a toxin-antitoxin formulation and was found to be 85 percent effective in preventing diphtheria. In the 1920s, the toxin was treated with formalin to produce toxoid, the toxicity of the preparation could be reduced while maintaining the immunogenic properties. In 1926, the alum-precipitated toxoid proved more effective, and by the mid-1940s diphtheria toxoid was being combined with tetanus toxoid and whole-cell pertussis vaccine to create the diphtheria-tetanus-pertussis (DTP) vaccine. Soon after, the DTP combination vaccine was adsorbed onto an aluminum salt and researchers noted the enhanced immunogenicity of the diphtheria and tetanus toxoid in the presence of pertussis vaccine and the aluminum salt.

  • Botulinum toxoid

In the early 1930s, a formalin-inactivated toxoid against botulinum neurotoxin was first tested in humans. Botulinum toxin (BoNT) production for the manufacture of toxoid vaccines is achieved by growing C. botulinum in fermenters using complex media. Culture medium consists of animal-derived or vegetable peptones, yeast extract, and glucose. Maximum toxin concentration can be attained after 24 h of fermentation. Nowadays, recombinant DNA technology has been employed to develop second-generation vaccines to prevent botulism. Recombinant subunit vaccines utilizing the receptor-binding domains of botulinum neurotoxin have been shown to be safe and efficacious in protecting animal models against BoNT.

  • C. Perfringens toxoid

C. Perfringens has been classified into seven toxinotypes (A–G) depending on the toxin they produce: alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), C. perfringens enterotoxin (CPE), or the necrotic enteritis beta-like toxin (NetB). Only type A (CPA), C (CPA and CPB), and F (CPA and CPE) are known to affect humans, whereas all of the toxinotypes have been shown to cause disease in animals. Animal types B, C, and D outbreaks can be prevented by immunization with crude toxoid or bacterin-toxoid vaccines, which have been proven to be effective in piglets, cattle, lambs, sheep, and goats. Similar to other pathogenic Clostridia, C. perfringens fermentation for vaccine production is poorly characterized. Purified toxins or the whole culture are chemically inactivated using formaldehyde, a time-consuming process that can lead to reduced immunogenicity. Therefore, the development, production and research of recombinant vaccine efficacy have been continuously strengthened.

The first whole-cell pertussis vaccines were licensed in the United States in 1914. These vaccines were suspensions of killed bacteria and were improved before being combined with diphtheria and tetanus toxoids to produce DTP vaccine. Owing to the reactogenicity of whole-cell vaccines, alternative vaccines were sought, and the first acellular vaccine was developed in Japan. These vaccines were composed of purified filamentous hemagglutin (FHA) and leukocytosispromoting factor hemagglutin. Currently, the acellular pertussis vaccine is only available in combination with diphtheria and tetanus in the United States.

Carrier protein

The hapten is a small chemical group that cannot cross-link the B-cell receptor and does not recruit T cells to help, so it cannot stimulate the antibody response in its free soluble form. When conjugated to the carrier protein, the hapten becomes immunogenic. The protein can carry multiple hapten groups which can now cross-link B-cell receptors and activate T cells through peptides derived from the carrier protein. Hapten-protein conjugates are crucial for generating high-titer and high-affinity antibodies. Toxoids can be used to couple haptens through any of the chemical reactions. They generate strong immunological responses in vivo.

Conjugate vaccines, typically composed of a pathogen-specific capsular (CPS) or O-antigen polysaccharide (O-PS) linked to an immunostimulatory protein carrier, are among the safest and most effective methods for preventing life-threatening bacterial infections. The conventional process to produce conjugate vaccines involves chemical conjugation of carrier proteins with polysaccharide antigens purified from large-scale cultures of pathogenic bacteria. There are five main carrier proteins used in vaccines today: tetanus toxoid, diphtheria toxoid, cross reactive material 197 (CRM197), N. meningitides outer membrane protein (OMP), and non-typeable H. influenza derived protein D (PD).

A typical process for production of a multivalent conjugate vaccine

Fig. 2 A typical process for production of a multivalent conjugate vaccine (Jessica O. Josefsberg and Barry Buckland. 2012)

Key advantages of the well-known recombinant subunit vaccine platforms are their safety, stability at 2−8 °C and facility to scale-up the production. Weak immunogenicity for small recombinant protein, requiring repeated vaccination. Most recombinant vaccines relied on macromolecular constructs as a way to increase the immunogenicity and on the use of potent adjuvants. The conjugation of the receptor binding domain (RBD) and TT led to a notable enhancement of the neutralizing response. The resulting vaccine candidate composed of an SARS-CoV-2 RBD-Tetanus toxoid conjugate, the first one developed in Latin America, has important advantages, such as (a) induction of strong IgG neutralizing antibody and specific T-cell responses, (b) the well-known safety record of this vaccine platform, (c) its stability at 2−8 °C allowing an effective distribution, and (d) both the expression and conjugation technologies’ ability to be adapted to existing production capacities available in many countries.

Conjugation of RBD with TT and representation of RBD2-TT and RBD6-TT

Fig. 3  Conjugation of RBD with TT and representation of RBD2-TT and RBD6-TT (Yury Valdes-Balbin, et al. 2021)

Human antitoxins

Antitoxins are life-saving drugs, but the way they are manufactured has not changed in more than 100 years. Almost all commercial antitoxins have been manufactured using serum from equines who have been hyperimmunised by repeated toxin injections. Animal sera have many safety disadvantages, including hypersensitivity, serum sickness and they also present the risk of transmitting viruses and other sources of disease between species. Toxoids are useful in the production of human antitoxins. Multiple doses of tetanus toxoid are used by many plasma centers in the United States for the development of highly immune persons for the production of human anti-tetanus immune globulin, which has replaced horse serum-type tetanus antitoxin in most of the developed world. Human antitoxins eliminate many of the practical drawbacks of equine-derived antitoxins, including avoiding the possibility of serum sickness or zoonotic disease transmission.

Creative diagnostics offers a range of high-quality formaldehyde inactivated native toxins such as tetanus toxoids, diphtheria toxoids, botulinum toxoids, C. difficile toxoids, B. pertussis toxoids and S. aureus toxoids. These toxoids are appropriate for use as antigens in development of antibodies and vaccine related research. If you are interested in finding out more about our toxoid products, please feel free to contact us.

References

  1. Nalin DR . Adverse Events Associated with Childhood Vaccines: Evidence Bearing on Causality. The Yale journal of biology and medicine, 1995, 67(5-6):281-282.
  2. Mueller JHand MillerPA. Production of diphtheria toxin of high potency (100 Lf) on a reproducible medium. The Journal of Immunology. 1941, 40:21-32.
  3. Nicolas EZ,Camila AO,Glenn AM, et al. Vaccine Production to Protect Animals Against Pathogenic Clostridia. Toxins. 2019, 11(9), 525.
  4. Pavimol A, RonnieHF,ZhangLF. Toxoid Vaccination Against Bacterial Infection Using Cell Membrane-Coated Nanoparticles. Bioconjugate Chemistry. 2018, 29(3): 604–612.
  5. Susumu K, Shinhachiro T, Hiroaki M, et al. Age-related decrease in frequencies of B-cell precursors and specific helper T cells involved in the IgG anti-tetanus toxoid antibody production in humans. Clinical Immunology and Immunopathology. 1982, 25 (1): 1-10.
  6. Oscar BT, Carl RA, Gary RM. Synthesis of Hapten-Protein Conjugate Vaccines with Reproducible Hapten Densities. Methods in Molecular Biology. 2016, 1403:695-710.

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