Select Toxins Research Articles

Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?

Is regulation preventing the development of therapeutics that may prevent future coronavirus pandemics?

Psychological Evaluations for the U.S. Army Biological Personnel Reliability Program

Abstract Given the inherent dangers associated with working in biodefense labs, coupled with the potential for insider threat, the Department of Defense and Federal Agencies are charged with determining the most suitable and reliable personnel for work in this high risk setting. A critical component to meeting this charge are Personnel Reliability Programs which aim to ensure the safety, security, and health of personnel who work with biological select agents and toxins. Behavioral health consultants can be an asset within an interdisciplinary approach for determining personnel suitability. The following perspective, on conducting psychological evaluations for the Army’s Biological Personnel Reliability Program, is intended to provide a framework for these complex exams along with highlighting relevant procedural, policy, and legal issues.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities.

ASM Public Affairs:ASM Comments on the List of Select Agents and Toxins

ASM Public Affairs:ASM Comments on the List of Select Agents and Toxins

Review of Restricted Experiment Requests, Division of Select Agents and Toxins, Centers for Disease Control and Prevention, 2006-2013.

The Centers for Disease Control and Prevention (CDC) Division of Select Agents and Toxins (DSAT) regulates laboratories that possess, use, or transfer select agents and toxins in the United States. DSAT also mitigates biosafety risks through the review of "restricted experiments," which under the select agent regulations are experiments that pose heightened biosafety risks. From January 2006 through December 2013, DSAT received 618 requests from 109 entities to perform potentially restricted experiments. Of these requests, 85% were determined not to meet the regulatory definition of a restricted experiment, while 15% of the requests met the definition of a restricted experiment. Of the 91 restricted experiments proposed, DSAT approved 31 (34%) requests because the biosafety conditions proposed were commensurate with the experiments' biosafety risk. All 31 approved restricted experiments were for work with select toxins. DSAT did not approve 60 restricted experiment requests due to potentially serious biosafety risks to public health and safety. All 60 denied restricted experiments proposed inserting drug resistance traits into select agents that could compromise the control of disease. The select agents and toxins associated most frequently with requests that met the regulatory definition of a restricted experiment are Shiga toxin (n = 16), Burkholderia mallei (n = 15), Botulinum neurotoxin (n = 14), and Brucella abortus (n = 14). In general, all restricted experiment decisions are determined on a case-by-case basis. This article describes the trends and characteristics of the data associated with restricted experiment requests among select agents that have an impact on public health and safety (HHS only agents) or both public health and safety and animal health or products (overlap agents). The information presented here, coupled with the information published in the restricted experiment guidance document ( www.selectagents.gov ), is intended to promote awareness among the research community of the type of experiments that meet the regulatory definition of a restricted experiment as well as to provide a greater understanding of the restricted experiment review process.

Implementing the Select Agent Legislation: Perfect Record or Wrong Metric?

The article “Transfer of Select Agents and Toxins: 2003-2013” by Shelby et al that appears in this issue describes in great detail the type and exact number of transfers of select agents, the day of the week packages were shipped, and the calendar days in transit. It also characterizes the “entities” involved in the transfers. The activities described are in response to the select agent regulations (SAR) promulgated in 19971 and 2003.2 The Centers for Disease Control's Division of Select Agents and Toxins3 (DSAT) and the US Department of Agriculture Select Agent Services4 make up the Federal Select Agent Program (FSAP), which approves and oversees transfer of agents that “pose a severe threat to public health and safety,” thereby “protecting public health and safety.”* Let's assume for the sake of this argument that the select agent regulations were brilliantly drafted, necessary, and sufficient. Shelby et al's article and the resources current and proposed to operate the select agent program, viewed in the shadows of an apparently increasing number of shipment errors in select agent labs, suggest that it might be time to revisit the implementation of the program.

Occupational Health and Personnel Suitability for Biological Research Programs

This article provides an outline of the federal government's occupational health and personnel suitability guidance to work with certain highly infectious and dangerous pathogens. It also includes the results of a survey conducted by Arizona State University regarding the individual requirements to work with these materials. Research shows that a limited number of tools are available to determine the necessary elements of pre-access personnel suitability assessments, ongoing personnel suitability assessments, and occupational health programs for personnel working with Tier 1 and non-Tier 1 Biological Select Agents and Toxins (BSAT). Self-reported answers from biosafety and biosecurity professionals regarding these issues are evaluated here.

Pathogen security-help or hindrance?

Events over the past 15 years have resulted in the promulgation of regulations in the United States to enhance biosecurity by restricting the access to pathogens and toxins (i.e., biological select agents and toxins [BSATs]), which pose a severe threat to human being, animal, or plant health or to animal or plant products, to qualified institutions, laboratories, and scientists. These regulations also reduce biosafety concerns by imposing specific requirements on laboratories working with BSATs. Furthermore, they provide a legal framework for prosecuting someone who possesses a BSAT illegally. With the implementation of these regulations has come discussion in the scientific community about the potential of these regulations to affect the cost of doing BSAT research, hamper research and international collaborations, or whether it would stop someone with a microbiological background from isolating many of the select agents from nature.

Differential neutralizing activities of a single domain camelid antibody (VHH) specific for ricin toxin's binding subunit (RTB).

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or “nanobody”) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1∶10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.

New Entity CDC Select Agent Registration Process—A Practical Guide to Lessons Learned

Entities planning to conduct work in the United States with biological select agents and toxins (BSAT) must be registered with either the Centers for Disease Control and Prevention (CDC) or the Ani...

Implementation of an Occupational Health and Medical Surveillance Program at National Biodefense Analysis and Countermeasures Center

Historically in the United States, occupational health and medical surveillance programs for biodefense laboratories that work with Biological Select Agents and Toxins (BSAT) or other infectious organisms are sponsored by the federal government (DoD, HHS, USDA, etc.) or state universities (Jahrling et al., 2009; Kortepeter et al., 2008; Risi et al., 2010). These programs are operated through the use of onsite occupational health clinics or local hospitals. The clinics employ occupational health nurses, infectious disease physicians, and other licensed medical health professionals who provide services to all employees at a laboratory facility. The National Biodefense Analysis and Countermeasures Center (NBACC) at Fort Detrick, Maryland, is a Department of Homeland Security National Laboratory that is operated as a Federally Funded Research and Development Center by the Battelle National Biodefense Institute (BNBI), a private company. As such, the establishment and implementation of an occupational health and medical surveillance program proved to be challenging for BNBI and the Health and Safety Office in particular. This article documents all the constituent parts of a functional, effective, and responsive occupational health and medical surveillance program for a government-owned, contractor-operated biodefense laboratory, as well as how NBACC collaborates with other National Interagency Biodefense Campus (NIBC) partners in this effort. The partners of the National Interagency Confederation for Biological Research (NICBR) are: U.S. Army Medical Research and Materiel Command; National Institute of Allergy and Infectious Diseases; National Cancer Institute, U.S. Department of Agriculture, Agricultural Research Service; Department of Homeland Security; Centers for Disease Control and Prevention; Naval Medical Research Center; and Food and Drug Administration.

Laboratory Decontamination of HHS-Listed and HHS/USDA Overlap Select Agents and Toxins

This review describes the susceptibilities of all currently HHS-listed and HHS/USDA Overlap select agents and toxins to 8 chemical and 2 physical decontaminants: alcohols, aldehydes, bleach/hypochl...

Implementation of a Personnel Reliability Program as a Facilitator of Biosafety and Biosecurity Culture in BSL-3 and BSL-4 Laboratories

In late 2010, the National Biodefense Analysis and Countermeasures Center (NBACC) implemented a Personnel Reliability Program (PRP) with the goal of enabling active participation by its staff to drive and improve the biosafety and biosecurity culture at the organization. A philosophical keystone for accomplishment of NBACC's scientific mission is simultaneous excellence in operations and outreach. Its personnel reliability program builds on this approach to: (1) enable and support a culture of responsibility based on human performance principles, (2) maintain compliance with regulations, and (3) address the risk associated with the insider threat. Recently, the Code of Federal Regulations (CFR) governing use and possession of biological select agents and toxins (BSAT) was amended to require a pre-access suitability assessment and ongoing evaluation for staff accessing Tier 1 BSAT. These 2 new requirements are in addition to the already required Federal Bureau of Investigation (FBI) Security Risk Assessment (SRA). Two years prior to the release of these guidelines, NBACC developed its PRP to supplement the SRA requirement as a means to empower personnel and foster an operational environment where any and all work with BSAT is conducted in a safe, secure, and reliable manner.

Neurologic Melioidosis in an Imported Pigtail Macaque (Macaca nemestrina)

Burkholderia pseudomallei is the cause of melioidosis in humans and other animals. Disease occurs predominately in Asia and Australia. It is rare in North America, and affected people and animals typically have a history of travel to (in human cases) or importation from (in animal cases) endemic areas. We describe the gross and histopathologic features and the microbiologic, molecular, and immunohistochemical diagnoses of a case of acute meningoencephalomyelitis and focal pneumonia caused by B. pseudomallei infection in a pigtail macaque that was imported from Indonesia to the United States for research purposes. This bacterium has been classified as a Tier 1 overlap select agent and toxin; therefore, recognition of pathologic features, along with accurate and timely confirmatory diagnostic testing, in naturally infected research animals is imperative to protect animals and personnel in the laboratory animal setting.

The Endurance Testing Process: A Methodology to Verify the Functionality of Biocontainment, Security, and Life Safety Systems in BSL-3 and BSL-4 Laboratories

Biocontainment facilities planning to possess or work with biological select agents and toxins (BSAT) require registration with the National Select Agent Registry. The registration process includes submission of documentation for review to either the Centers for Disease Control and Prevention (CDC) Select Agent Program or the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Services, followed by a site inspection. The process serves to assess whether an entity meets requirements to safely and securely conduct work with BSAT. Part of the assessment focuses on the facility itself with regards to design, construction and engineering. Testing and verifying that facility systems integral to biocontainment and overall safety and security are functional and reliable is an essential prerequisite. The National Biodefense Analysis and Countermeasures Center (NBACC) developed a process of endurance testing to verify the facility could be operated in a safe and secure manner. Information prese...

The Impact of Regulations, Safety Considerations and Physical Limitations on Research Progress at Maximum Biocontainment

We describe herein, limitations on research at biosafety level 4 (BSL-4) containment laboratories, with regard to biosecurity regulations, safety considerations, research space limitations, and physical constraints in executing experimental procedures. These limitations can severely impact the number of collaborations and size of research projects investigating microbial pathogens of biodefense concern. Acquisition, use, storage, and transfer of biological select agents and toxins (BSAT) are highly regulated due to their potential to pose a severe threat to public health and safety. All federal, state, city, and local regulations must be followed to obtain and maintain registration for the institution to conduct research involving BSAT. These include initial screening and continuous monitoring of personnel, controlled access to containment laboratories, accurate and current BSAT inventory records. Safety considerations are paramount in BSL-4 containment laboratories while considering the types of research tools, workflow and time required for conducting both in vivo and in vitro experiments in limited space. Required use of a positive-pressure encapsulating suit imposes tremendous physical limitations on the researcher. Successful mitigation of these constraints requires additional time, effort, good communication, and creative solutions. Test and evaluation of novel vaccines and therapeutics conducted under good laboratory practice (GLP) conditions for FDA approval are prioritized and frequently share the same physical space with important ongoing basic research studies. The possibilities and limitations of biomedical research involving microbial pathogens of biodefense concern in BSL-4 containment laboratories are explored in this review.

Monitoring Select Agent Theft, Loss and Release Reports in the United States—2004–2010

The APHIS/CDC Form 3, Report of Theft, Loss or Release of Select Agents and Toxins (TLR incident report) is the mechanism by which the theft, loss or release of a biological select agent and toxin ...

Medical Countermeasures for Biothreat Agents: in vivo Studies and Animal Models

Biothreat agents are prioritized in terms of their threat potentials by taking several factors into account, such as inhalation route of infection / intoxication, infectious dose or toxicity, stability in the environment, and availability of therapeutics / prophylaxis. Most of these factors are intrinsic features of the given pathogen or toxin and are not under our control to modulate. However, pre-exposure prophylaxis and post exposure therapeutics are the criteria that intensive research can handle and dislodge a particular organism from the Select Agent and Toxin List (SATL). Our ability, to impart immunity to human population against an infectious agent and/or treat the disease, substantially mitigates a potential threat and renders the agent obnoxious from bioterrorism or warfare viewpoint. For instance, Clostridium tetani and its neurotoxin are equivalent to Clostridium botulinum at least in terms of the lethality of the neurotoxins but the former is not considered a potential select agent for the availability of prophylactic measures and immunization regimen. The problems associated with pre-exposure prophylaxis for threat agents is confounded by several factors including the long list of select agents and the uncertainty of use for a particular agent. For the agents of public health importance, we are aware of the endemecity of natural disease outbreaks, but it is difficult to predict which agent will be used in a bioterror or BW attack. Theoretically, a pre-exposure prophylaxis in biothreat scenario requires immunization against all the agents or least against those posing potential threat. Assuming that we have developed vaccines for each of the threat agents, the vaccination of even a vulnerable population for this wide array of pathogens is a magnanimous task.

Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.