Select Agents and Toxins Exclusions

Nontoxic HHS toxins (Section 73.3 (d)(2))

Nontoxic select toxins are automatically excluded from the requirements of the select agent regulations and do not require prior review from the federal select agent program. Nucleic acids that encode for the nontoxic form of the toxin proteins listed below are also excluded from the requirements of the select agent regulations              


BOTULINUM NEUROTOXIN

Note: nucleic acids that encode for the less potent or toxic form of the specific Botulinum neurotoxin proteins listed below are also excluded from the requirements of the select agent regulations

  • Catalytically inactive Botulinum neurotoxin (ciBoNT) B, C, E, F (effective March 23, 2016)
    Injection of up to 8ug per mouse did not result in death or any signs of botulism and therefore, represent nontoxic proteins.

    Reference(s):
    1. Webb RP1, Smith TJ, Wright P, Brown J, Smith LA.Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit vaccines against toxin subtypes A1, A2, and A3. Vaccine. 2009 Jul 16;27(33):4490-7. doi: 10.1016/j.vaccine.2009.05.030. Epub 2009 May 28.
    2. Mukherjee J1, Tremblay JM, Leysath CE, Ofori K, Baldwin K, Feng X, Bedenice D, Webb RP, Wright PM, Smith LA, Tzipori S, Shoemaker CB. A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS One. 2012;7(1):e29941. doi: 10.1371/journal.pone.0029941. Epub 2012 Jan 6.
    3. Smith, LA, Webb, RP, Smith, TJ, Wright, PM, Guernieri RL, Brown, JL and Skerry, JC. Production and Immunological Assessment of Recombinant, Catalytically Inactive Clostridium botulinum Neurotoxin Holoproteins as Potential Vaccine Candidates. Poster presentation at Toxins 2015 meeting, Lisbon Portugal January 14-17 and at the Interagency Botulism Research Coordinating Committee (IBRCC), Frederick, MD, October 25-28

  • Botulinum neurotoxin type C atoxic derivative (BoNT/C ad) (effective December 23, 2014)
    Three mutations were introduced in the light chain construct (E446>A; H449>G; Y591>A) of a modified BoNT/C ad, resulting in a modified protein demonstrated to be significantly less potent or toxic than the wild-type. The three original amino acids (E446; H449; Y591) are conserved among BoNT serotypes and represent part of the catalytic core of the light chain metalloprotease, responsible for cleavage of the substrate(s).  Data (unpublished) showed that cleavage of substrates syntaxin-1 and SNAP-25 were undetected in neuronal cell based assays, when hippocampal neuronal cultures were exposed to 500 nM BoNT/C ad. The non-catalytic protein was also tested for in vivo toxicity in a mouse bioassay. The LD50 of the BoNT/C ad is > 100 ug/protein per ~20 g mice, which is ~10,000,000-fold more than the LD50 of wild type BoNT/C (data unpublished).

  • Fusion proteins of the heavy-chain domain of BoNT/translocation domain of diphtheria toxin (effective 07-28-2011)
    Fusion proteins consisting of the heavy-chain domain of BoNT and the translocation domain of diphtheria toxin (no catalytic domain); however, the reconstitution of the BoNT holotoxin would be considered a select toxin.

    Reference(s):
    1. Ho M, Chang LH, Pires-Alves M, Thyagarajan B, Bloom JE, Gu Z, Aberle KK, Teymorian SA, Bannai Y, Johnson SC, McArdle JJ,Wilson BA. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting. Protein Engineering, Design and Selection. 2011 Mar; 24(3):247-53.

  • Recombinant catalytically-inactive botulinum A1 holoprotein (ciBoNT/A1 HP) (effective May 7, 2010)
    Active site mutations were created at h223>a and h227>a positions, which renders the catalytic domain unable to coordinate the zinc atom needed for catalytic activity, and position e224>a, a mutation which renders the active site unable to bind the water molecule needed for hydrolysis. Full length toxin was confirmed to be catalytically inactive using immunological assays. Purified native BoNT/A1 toxin at 69nM yielded an average specific activity at 15.8 µmol/min/mg, while the ciBoNT/A1 HP, at the same concentration, failed to produce any detectable SNAP-25 peptide cleavage products. Increasing the ciBoNT/A1 HP concentration from 69nM to 104nM failed to elicit substrate cleavage, indicating the ciBoNT/A1 HP had no detectable proteolytic activity at the levels tested (data not published). The catalytically inactive BoNT/A1 showed no toxicity in mice (data not published).

    Reference(s):
    1. 1. Schmidt JJ, Bostian KA.Endoproteinase activity of type A botulinum neurotoxin: substrate requirements and activation by serum albumin. J Protein Chem. 1997 Jan;16(1):19-26.
    2. 2. Zhou L, de Paiva A, Liu D, Aoki R, Dolly JO. Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain. Biochemistry. 1995 Nov 21;34(46):15175-81.

  • BoNT purified protein (BoNT/A1 atoxic derivative, ad, E224A/Y366A) (effective 07-22-2009)
    BoNT purified protein (BoNT/A1 atoxic derivative, ad, E224A/Y366A) that has been expressed from recombinant DNA constructs is non-catalytic and non-toxic as a result of a double mutation introduced into the region of the gene encoding the light chain.

  • Recombinant Botulinum neurotoxin serotype A (R362A, Y365F) (effective 03-28-2006)
    Recombinant Botulinum neurotoxin serotype A (R362A, Y365F), termed BoNT/A(RY), is a product of two amino acid substitutions engineered into the light chain of Botulinum neurotoxin serotype A (BoNT/A). Arg362Ala (three base substitution) and Tyr365Phe (two base substitution) yields a protein that does not cleave SNAP25 (the mutated toxin is >100-fold less catalytic than native protein). One microgram of recombinant Botulinum neurotoxin serotype A BoNT/A (RY) is not toxic in the mouse model of BoNT intoxication.

    Reference(s):
    1. Rossetto O, Caccin P, Rigoni M, Tonello F, Bortoletto N, Stevens RC, Montecucco C. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity. Toxicon. 2001 Aug; 39(8):1151-9.
    2. Barbieri JT, Collier RJ. Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli. Infect Immun. 1987 Jul; 55(7):1647-51.

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CONOTOXINS
  • Conotoxins (non-short, paralytic alpha conotoxins; effective 12-4-2012)
    Based upon available experimental evidence, most known conotoxins (i.e.,"conopeptides") do not possess sufficient acute toxicity to pose a significant public health threat, and many are employed as useful research tools or potential human therapeutics. However, currently available data demonstrate that the sub-class of conotoxins generally called "short, paralytic alpha conotoxins," exemplified by α-conotoxin GI and α-conotoxin MI, do possess sufficient acute toxicity by multiple routes of exposure, biophysical stability, ease of synthesis, and availability. The conotoxins that remain on the HHS list will be limited to the short, paralytic alpha conotoxins containing the following amino acid sequence X1 CCX2 PACGX3 X4 X5 X6 CX7, whereas:

    • The consensus sequence includes known toxins α-MI and α-GI (shown above) as well as α-GIA, Ac1.1a, α-CnIA, α-CnIB;
    • C = Cysteine residues are all present as disulfides, with the 1st and 3rd Cysteine, and the 2nd and 4th Cysteine forming specific disulfide bridges;
    • X1 = any amino acid(s) or Des-X;
    • X2 = Asparagine or Histidine;
    • P = Proline;
    • A = Alanine;
    • G = Glycine;
    • X3 = Arginine or Lysine;
    • X4 = Asparagine, Histidine, Lysine, Arginine, Tyrosine, Phenylalanine or Tryptophan;
    • X5 = Tyrosine, Phenylalanine, or Tryptophan;
    • X6 = Serine, Threonine, Glutamate, Aspartate, Glutamine, or Asparagine;
    • X7 = Any amino acid(s) or Des X;
    • "Des X" = "an amino acid does not have to be present at this position." For example if a peptide sequence were XCCHPA then the related peptide CCHPA would be designated as Des-X.

    The short, paralytic alpha conotoxins containing the following amino acid sequence X1 CCX2 PACGX3 X4 X5 X 6 CX 7 will be considered a select toxin if the total amount (all forms) under the control of a principal investigator, treating physician or veterinarian, or commercial manufacturer or distributor exceeds 100 mg at any time.

    Reference(s):
    1. Favreau P, Krimm I, Le Gall F, Bobenrieth MJ, Lamthanh H, Bouet F, Servent D, Molgo J, Ménez A, Letourneux Y, Lancelin JM. Biochemical characterization and nuclear magnetic resonance structure of novel α -conotoxins isolated from the venom of Conus consors. Biochemistry. 1999 May 11; 38(19):6317-26.
    2. Groebe DR, Dumm JM, Levitan ES, Abramson SN. alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors. Mol Pharmacol. 1995 Jul; 48(1):105-11.
    3. Groebe DR, Gray WR, Abramson SN. Determinants involved in the affinity of α -conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors. Biochemistry. 1997 May 27; 36(21):6469-74.
    4. Liu L, Chew G, Hawrot E, Chi C, Wang C. Two potent alpha3/5 conotoxins from piscivorous Conus achatinus. Acta Biochim Biophys Sin (Shanghai). 2007 Jun; 39(6):438-44.
    5. Stiles BG. Acetylcholine receptor binding-characteristics of snake and cone snail venom postsynaptic neurotoxins: further studies with a non-radiological assay. Toxicon. 1993 Jul; 31(7):825-34.

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STAPHYLOCOCCAL ENTEROTOXINS (SE)

  • SEA triple mutant (L48R, D70R, and Y92A) (effective: January 16, 2014)
    It was shown that a single-site mutation, Y92A, in SE type A retained only 10% MHC-II binding. Two additional mutations, L48R and D70R, reduced binding to 1%. The biological activity of the triple mutant in cellular assays was highly attenuated. Human T-cell recognition by the mutant SEA was reduced a million fold (106) in comparison with that by the wild-type SEA.

  • SEB triple mutant (L45R, Y89A, Y94A) (effective: January 16, 2014)
    Studies of SE type B triple mutant produced in E. coli cells demonstrated a lack of super-antigen activity, using human, primate and pig leukocyte cultures. The results of immunization of non-human primates and pigs with this mutant SEB in research of efficiency of the protein as anti-SEB vaccine confirmed its non-toxic status. The triple mutant of SEB was expressed in transgenic soybean seeds, and lack of toxicity of the soybean-derived mutant SEB was shown.

  • SEC double mutant (N23A and Y94A) (effective: January 16, 2014)
    Absence of toxicity of a single mutant SE type C (N23 important for binding to TCR had been replaced with A23) and a double mutant SEC (N23 was replaced with A23 and Y94, important for binding to MHC-II, was substituted with A94) was shown in experiments on mice in efficiency studies of these mutants for protection against S. aureus infection.

  • Reference(s):
    1. Ulrich R. G., Olson M. A., and Bavari S. Development of engineered vaccines effective against structurally related bacterial superantigens. Vaccine, 16, 1857-1864, 1998.
    2. Bavari S., Dyas B., and Ulrich R. G. Superantigen vaccines: A comparative study of genetically attenuated receptor-binding mutants of staphylococcal enterotoxin A. J. Infect. Dis. 174, 338-345, 1996.
    3. Krupka H. I., Segelke B. W., Ulrich R. G., Ringhofer S., Knapp M., and Rupp B. Structural basis for abrogated binding between staphylococcal enterotoxin A superantigen vaccine and MHC-IIa. Prot. Sci. 11, 642-651, 2002.
    4. Boles J. W., Pitt M. L., LeClaire R. D., Gibbs P. H., Torres E., Dyas B., Ulrich R. G., and Bavari S. Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity. Clin. Immunol. 108, 51-59, 2003.
    5. Inskeep T. K., Stahl C., Odle J., Oakes J., Hudson L., Bost K. L., and Piller K. J., Oral vaccine formulations stimulate mucosal and systemic antibody response against staphylococcal enterotoxin B in a piglet model. Clin.Vaccine Immunol. 17, 1163-1169, 2010.
    6. Hudson L. C., Seabolt B. S., Odle J., Bost K. L., Stahl C. H., and Piller K. J., Sublethal staphylococcal enterotoxin B challenge model in pigs to evaluate protection following immunization with a soybean-derived vaccine. Clin.Vaccine Immunol. 20, 24-32, 2013
    7. Hu D.-L., Cui J.-C. Omoe K., Sashiami H., Yokomizo Y., Shinagawa K., and Nakane A. A mutant of staphylococcal enterotoxin C devoid of bacterial superanigenic activity elicits a Th2 immune response for protection against Staphylococcus aureus infection. Infect. Immun. 73, 174-180, 2005.
    8. Hu D.-L., Omoe K., Narita K., Cui J.-C., Shinagawa K., and Nakane A. Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection, Microbes Infect. 8, 2841-2848, 2006.

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