Principal Investigator: James E. Keller, PhD
Office / Division / Lab: OVRR / DBPAP / LRSP
We are establishing an integrated research program that includes the study of diphtheria, tetanus, and botulism toxoid vaccines. Toxoid vaccines are made by treating toxins produced by bacteria with heat or chemicals to render them harmless but still able to trigger protective immune system responses. Like the native (natural) toxins themselves, toxoids are composed of two proteins, called A and B.
While we are currently focused on botulism, many practical aspects of this work readily pertain to diphtheria and tetanus toxoid research. The lethal effects of each infection are caused by toxic proteins made by the corresponding disease-causing organisms (Clostridium tetani, Corynebacterium diphtheriae, or Clostridium botulinum). The vaccine to each is derived from a chemically detoxified toxin (toxoid) that is nontoxic but similar enough to the original toxin to retain the ability to be antigenic, that is, to trigger immune responses. The immune system response to these toxoids creates a long-term "memory" among specific antibody-producing cells that will subsequently respond to real toxins by producing protective antibodies against them.
Tetanus and diphtheria toxoids have been used as vaccines in the US for more than 70 years and are in about 25% of all vaccines licensed by the FDA. Similar to tetanus and diphtheria toxoids, botulinum toxoids trigger production of antibodies that neutralize (prevent killing action) the real botulinum toxin. However, there are currently no licensed vaccines to prevent botulism available in the US.
Importantly, all three toxoids share certain similarities that enable the use of similar manufacturing and testing techniques for these different vaccines. The most critical part of manufacturing is the design and use of effective test methods that ensure the manufacturing process preserves the antigenicity or the critical qualities needed to produce toxin neutralizing antibodies for each vaccine. Such tests have been available for tetanus and diphtheria vaccines for many decades, but are not available for botulism-based vaccines.
In response to this need, our laboratory developed tests that measure the antigenic similarity between botulinum toxoid A and B antigens and native botulinum A and B toxin antigens. Our studies have given us important new insights into the antigenic quality of these toxins and toxoids that neither industry nor academic laboratories previously provided. Specifically, we showed that out of a variety of botulinum toxoid antigens tested, those most similar to the native toxins elicit the highest protective immunity in animal studies.
Since the effectiveness of a toxoid vaccine depends on how closely the toxoid resembles the native toxin, our assays can help to identify the best botulinum vaccine candidate among several competing vaccine candidates. Furthermore, these new assays can help to screen competing vaccines that target diseases linked to toxins such as anthrax and ricin. Such screening will be critical because exposure to these toxins is rare, making it impossible to collect sufficient clinical data on the effectiveness of different vaccines to protect against them.
In the case of botulinum vaccine products, test methods that we develop can be used during the development and manufacture of products to determine how consistent they are from one batch to another. Therefore, our research will help us to ascertain the safety and effectiveness of such products and thus enhance our ability to regulate potential new vaccine products.
In the 1920s a reliable and simple test method was developed to measure the antigenic content of diphtheria toxin. By mixing a sample of toxin with a reference antitoxin, the assay measured the quantity of antibody needed to precipitate the toxin. This relatively fast, in vitro method allowed scientists to quantify toxin antigens even after toxicity had been eliminated by chemical treatment, heat or other means. Using the test, scientists were able to perform controlled chemical detoxification of diphtheria toxin that preserved its antigenicity, which enabled the development of a human diphtheria vaccine. A few years later this technique was adapted to measure tetanus toxin antigenicity and synthesize tetanus toxoids.
While both assays continue to be used in the manufacture of tetanus and diphtheria vaccines, these assays require large quantities of reagents. Therefore, our initial goal was to develop two antigenicity assays for botulinum A and B toxoids that would require relatively small volumes of reagents. This required creating new toxoids to the A & B botulinum neurotoxins and new antitoxins to each. Our central hypothesis was that an ideal toxoid is structurally indistinguishable from the parent toxin yet has little or no toxicity. Although this concept is intuitively straightforward, the botulinum field has never tried to compare the antigenicity of various vaccine candidates with the native toxin(s).
Our new botulinum antigenicity assays are modified inhibition ELISAs in which 96-well plates are coated with active neurotoxin. Dilutions of soluble toxoid are added to the toxin-coated wells, followed by a standard reference antibody made in this laboratory that binds to either toxoid or toxin. The assay quantifies the relative binding of antibody to toxoid or toxin, thus measuring their antigenic similarities. The key to the success of this test is our use of moderately detoxified toxins to generate high quality mouse antiserum. We used the antiserum to design an early version of the inhibition ELISA, which enabled us to evaluate and refine toxoid synthesis. The two projects proceeded cyclically, supporting the development of each other. We used the best A & B toxoids to generate two large pools of rabbit antisera, both of which replaced mouse antisera as the standard reagents used in the ELISA assays.
We have achieved two significant accomplishments thus far: 1) the final in-house ELISAs successfully quantify the antigenic content of each toxoid using relatively small quantities of reagents; and 2) the final in-house A & B toxoids are far superior to commercially available toxoids.
Horses were subsequently immunized with the new toxoids to generate equine antitoxins (anti-A and anti-B) that we are now testing as replacement potency standards under CFR 610.20(a). We continue to use the toxoids, antisera, and ELISAs to develop additional in vitro methods to measure antitoxin potency and protective immunity.
Biologicals 2012 Jul;40(4):240-6
New equine antitoxins to botulinum neurotoxins serotypes A and B.
Li D, Mattoo P, Keller JE
Procedia Vaccinol 2011;5:47-59
Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions
Isbrucker R, Levis R, Casey W, McFarland R, Schmitt M, Arciniega J, Descamps J, Finn T, Hendriksen C, Horiuchi Y, Keller J, Kojima H, Sesardic D, Stickings P, Johnson NW, Allen D
Procedia Vaccinol 2011;5(1):33-46
Improving animal welfare and reducing animal use for human vaccine potency testing: state of the science and future directions
Casey W, Schmitt M, McFarland R, Isbrucker R, Levis R, Arciniega J, Descamps J, Finn T, Hendriksen C, Horiuchi Y, Keller J, Kojima H, Sesardic D, Stickings P, Johnson NW, Lipscomb E, Allen D
Clin Vaccine Immunol 2008 Sep;15(9):1374-9
Characterization of new formalin-detoxified botulinum neurotoxin toxoids.
Infect Immun 2006 Oct;74(10):5617-24
Comparison of extracellular and intracellular potency of botulinum neurotoxins.
Cai F, Adrion CB, Keller JE
Recovery from botulinum neurotoxin poisoning in vivo.
Biochemistry 2004 Mar 2;43(8):2209-16
Role of metals in the biological activity of Clostridium botulinum neurotoxins.
Eswaramoorthy S, Kumaran D, Keller J, Swaminathan S
Biochemistry 2004 Jan 20;43(2):526-32
Uptake of Botulinum Neurotoxin into Cultured Neurons.
Keller JE, Cai F, Neale EA