TY - JOUR
AU - Lester, Patrick, A
AB - Abstract Research using laboratory animals has been revolutionized by the creation of humanized animal models, which are immunodeficient animals engrafted with human cells, tissues, or organs. These animal models provide the research community a unique and promising opportunity to mimic a wide variety of disease conditions in humans, from infectious disease to cancer. A vast majority of these models are humanized mice like those injected with human CD34+ hematopoietic stem cells and patient-derived xenografts. With this technology comes the need for the animal research enterprise to understand the inherent and potential risks, such as exposure to bloodborne pathogens, associated with the model development and research applications. Here, we review existing humanized animal models and provide recommendations for their safe use based on regulatory framework and literature. A risk assessment program—from handling the human material to its administration to animals and animal housing—is a necessary initial step in mitigating risks associated with the use of humanized animals in research. Ultimately, establishing institutional policies and guidelines to ensure personnel safety is a legal and ethical responsibility of the research institution as part of the occupational health and safety program and overall animal care and use program. animal models, biosafety, bloodborne pathogens, humanized animals, occupational health and safety, regulations Introduction Humanized animal models have become increasingly important and useful scientific translational biomedical tools to understand and elucidate the pathophysiology and mechanisms of human disease, including cancer, infectious disease, hematology, immune-mediated pathology, regenerative medicine, and cellular functions of human disease. As animal models become more closely aligned with human disease, the risk to human personnel may also increase. As a result, additional safety factors including engineering, personal protective equipment (PPE) and standard guidance policies should be applied. This manuscript first describes the history of humanized animal model development, including commonly utilized models and their translational scientific applications. Relevant laws and regulations, guidance publications, and components of an occupational health and safety program (OHSP) will then be discussed. Lastly, a risk assessment program and recommendations to ensure personnel safety when working with human materials and humanized animals will be presented. Mouse Humanized Models History In general, humanized animal models can be described as immunodeficient animal models engrafted with human cells, tissues, or organs.1–3 These models closely replicate human physiologic, cellular, and immune system functions. Most commonly, immunodeficient mice or those expressing human transgenes are engrafted with human progenitor cells, primary hematopoietic cells, tissues, or organs (eg, immune system, skin, reproductive, or digestive system tissues) that generate human functional tissues or systems (eg, immune system). The most common humanized models used in research include immunodeficient mice engrafted with a human immune system, which will be the primary focus of this article. Historical discovery timelines and events surrounding the development and utilization of immunodeficient mice and their role in humanized research have been published.1,2,4,5 The development of humanized animals first arose from the 1966 discovery of an immune deficient athymic nude mouse phenotype; the nude phenotype resulted from diminished keratin and brittle hair,6 caused by a mutation in the transcription factor Foxn1 gene, which affects multiple downstream targets.7 Homozygous athymic nude mice lack a thymus and have severe defects in T cell maturation and function, including a reduced ability to mount a T-cell-dependent adaptive immune response. These characteristics make athymic nude mice a popular research tool for studying allografts, cancer biology, and xenografts, including patient-derived xenografts. However, athymic nude mice may develop T cell markers as they age and possess an intact and enhanced innate immune system including natural killer cells and macrophages.8,9 These factors reduce the athymic nude mouse’s ability to establish and engraft a fully functional humanized immune system model.10 Approximately 20 years after the discovery and characterization of the athymic nude mouse, a severe combined immunodeficiency mutation (scid) was described in a C.B-17 mouse strain. The homozygous scid phenotype lacks both mature T and B lymphocytes with loss of cell-mediated and humoral immunity.11,12 A nonsense mutation in the protein kinase, DNA activated, catalytic polypeptide Prkdcscid gene was shown to disrupt the catalytic subunit of a DNA-dependent protein kinase responsible for V(D)J recombination of antigen receptors, leading to a severe combined immunodeficiency phenotype and enhanced sensitivity to ionizing radiation.13–17 However, scid mice have demonstrated strain dependent antigen recombination within 3 to 9 months of age, secondary to a low frequency of V(D)J short section recombination described as “leaky” or “leakiness.”18,19 Similar to the athymic nude mouse, scid mice also possess an active innate immunity with natural killer cell, macrophage, and compliment activity.20 Allografts and xenografts have been implanted in scid mice in the absence of severe cell-mediated rejection.21–23 Conversely, due to antigen leakiness, innate immunity and sensitivity to radiation, scid mice humanized with peripheral blood leukocytes, bone marrow cells, or human tissues are associated with low engraftment rates.24,25 To overcome the inherent innate immunity in scid mice and to improve engraftment with human lymphoblastic hematopoietic cells, scid mice were backcrossed onto a non-obese-diabetic NOD mouse strain known for its immune system defects, including lack of complement C5 and reduced natural killer cell, dendritic, and myeloid function.26 Due to improved engraftment rates, NOD/SCID mice have been used for decades to study human immune system function and related diseases, yet long-term studies with NOD/SCID mice are problematic due to incomplete human immune system development due to impaired T cell activation, a high incidence of thymic lymphoma, shorter lifespan of 6 to 9 months, and murine immune leakiness, although less than is observed with scid mice.1,27,28 Xenogenic graft versus host disease (GVHD) secondary to activated human T cells against mouse tissues may also increase murine model morbidity and mortality. The pivotal breakthrough for humanized mouse models was the targeted mutation of the murine interleukin-2 receptor subunit gamma chain, Il2rγ, (ie, Il2 receptor common gamma chain) encoding a signal transduction transmembrane receptor subunit shared by receptors IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.29 Back-crossing the Il2rγ−/− mice with NOD-scid double homozygous mice established the NOD-scid/gamma null models, namely NOD/LtSz-scid Il2rγ−/− (NSG)30 and NOD/Shi-scid Il2rγ−/− (NOG),31 both devoid of mature T and B cells and natural killer cell activity, with very low antigenic leakiness with age and defective dendritic cells and macrophages.2,29,30,32 The NSG mouse has established its utility as an efficacious humanized mouse model, in particular due to its longer average lifespan (approximately 2 years compared with 5–6 months with NOD-scid mice), most likely resulting from reduced incidence of IL-2-dependent thymic lymphoma.30,31,33–35 Pearson et al. described the development of a NOD model crossed with targeted mutations in Il2rγ−/− and the recombination activating gene Rag-/- to create a NOD-Rag-/-Il2rγ−/− (NRG) model, which provided enhanced resistance to ionizing radiation versus the NSG mouse model.36 In addition to the NSG and NOG models, the BRG model was developed by backcrossing Il2rγ−/− mice to BALB/c recombination activating gene Rag2 deficient mice, BALB/c-Rag2−/−Il2rγ−/−.37 The NOG mouse model has a truncated cytoplasmic domain of the IL-2 receptor gamma chain that can bind cytokines, yet signaling is nullified compared with an absent gamma chain that cannot bind cytokines nor process signaling in NSG and BRG mice.38 In general, Il2rγ−/− mice demonstrate minimal lymphatic tissue or node formation due to disruption in T cell maturation and signaling. Differences exist between the NOG, NSG, NRG, and BRG models in regards to engraftment using a humanized immune system. Ito et al. reported the following order for humanized tissue and cell engraftment with NSG = NOG > NRG > BRG > NOD-scid.4,31,39 Furthermore, due to enhanced T cell support, engraftment rates may be higher in younger (up to 3-4 weeks) NSG mice compared with older NSG mice associated with limited lymphoid tissue or thymus development.30,33,40 However, all of these immunodeficient models have been shown to be useful models for humanized immune system research.35,41,42 Model Development There are 3 methodologies generally utilized for engrafting human immune systems into immunodeficient mice based upon current literature. With slight differences, the humanized-peripheral blood lymphocytes-SCID, humanized-scid-repopulating cell-SCID, and bone marrow, liver, thymus (BLT) humanized models allow for the engraftment of primary human immune system components including T cells, B cells, natural killer lymphocytes, dendritic cells, and macrophages, yet engraftment and development of erythroid cells, platelets, and granulocytes is usually less efficient.2 Immunodeficient mice used to create humanized models often receive sublethal doses of irradiation prior to injection of CD34+ cells to improve human immune system engraftment. The humanized-peripheral blood lymphocytes-SCID model is created via injection of human peripheral blood, spleen, or lymph node cells with rapid engraftment of CD34+ cells within 1 week, allowing for the study of mature T lymphocytes. The humanized-scid-repopulating cell-SCID model incorporates engraftment of intravenous or intraosseous injection of human CD34+ hematopoietic stem cells from multiple sources (eg, bone marrow, umbilical cord blood, or fetal liver), producing a functional immune system. The BLT model, which generates improved human adaptive and innate immune responses, utilizes fetal bone marrow, fetal liver, and fetal thymus. Fetal liver and thymus are implanted in the subrenal capsule, followed by sublethal radiation and intravenous injection of autologous fetal hematopoietic stem cells.1,38,40,43 The BLT model allows for maturation of human T cells in human thymic tissue with subsequent improvements in T cell development, enhanced interactions between human T and B cells, and improved immunoglobin isotype switching.2 The BLT model also demonstrates human mucosal immune system functionality, making it a useful tool for human immunodeficiency virus (HIV) research. The development of humanized animal models is a complex process requiring immunocompromised animals, human cells or tissues, irradiation, surgical expertise, and potential lengthy intervals until engraftment.41 Multiple safety processes and procedures are paramount when working with human tissue or disease agents, especially in models that can promote their dissemination or potentially expose personnel. Limitations The development of lymph node tissue architecture and germinal centers in common immunodeficient models used to create humanized immune model systems (eg, NSG, NOD, NGR, or BRG) is inconsistent due to lack of T cell development and B cell maturation secondary to a targeted mutation at the interleukin-2 receptor subunit gamma (cytokine receptor common gamma-chain) or Il2rγ−/−.40 Human and murine immune systems demonstrate multiple differences in major histocompatibility complexes (MHCs) (eg, murine MHC class I and II vs human leukocyte antigen, or HLA), which may limit recognition of antigenic peptides between host and species of engraftment. One approach to overcome these differences is to insert HLA transgenes autologous to HLA-matched human stem cells and/or create targeted null mutations in murine MHC molecules in immunodeficient models prior to humanization. Cytokine signaling normally involved with human T and B cell maturation, immune system functionality, and development of myeloid and erythroid lineages may be diminished and inconsistent unless human cytokines and growth factors (eg, IL-3, GM-CSF, or thrombopoietin) are genetically inserted or externally administered to humanized immunodeficient mouse models.40 Due to mechanistic immune system and cell signaling differences between human and mouse species and their complexity, humanized mouse models may demonstrate deficiencies in primary T cell development and maturation, reduced numbers of memory T cells, limited immunoglobulin isotype class switching with IgM predominating, and reduced mucosal immunity organization.2,44 Maturation and functionality is improved with the BLT humanized model, which utilizes fetal thymic and liver tissue to promote human-specific T cell education and maturation processes, yet additional approaches to reduce murine innate immunity, improve human innate immunity, and promote human adaptive immunity are needed and warranted.40 These methodologies have been previously reviewed.2,3,32,38,40,45,46 In addition to deficiencies in complete immune system functionality and maturation, humanized immunodeficient mouse models are prone to wasting disease or development of xenogenic GVHD, most likely due to reactivity to murine MHC class I and II molecules and residual murine innate immunity. The rate to develop xenogenic GVHD varies between the NSG and BRG immunodeficient models used for creating a humanized murine model, with NSG mice experiencing xenogenic GVHD at a faster rate than BRG mice.47 One solution is the use of targeted mutations in murine MHC I and II genes in immunodeficient mouse models that may reduce the incidence or development of xenogenic GVHD.40 Non-Mouse Immunodeficient Models In addition to humanized mouse models, immunodeficient rabbits and swine have recently been produced through genomic editing or natural breeding as research models for allografts, xenotransplantation, and regenerative medicine. The need for nonmurine or larger immunodeficient animal models that may support human cellular or tissue engraftment is warranted due to research that may require device implant, tissue-engineered constructs, transplantation, or collection of larger tissue, blood, or target sample volumes. Utilizing CRISPR/Cas9 technology for single-gene and multigene editing, Yan et al.48 and Song et al.49 produced immunodeficient rabbits with deletions in FOXN1, RAG2, IL2RG, and PKRDC genes, including multigene combinations. Additional research with these models is needed to demonstrate if immunodeficient rabbits can be successfully engrafted with human hematopoietic stem cells or fetal tissues. If necessary, additional genetic editing to enhance engraftment, improve immune system functionality, and reduce rejection rates of human immune systems in rabbits and in other species should be feasible and efficient with the use of CRISPR/Cas9 technology. Pigs have genetic, anatomical, and physiologic similarities to humans. Recently, severe combined immune-deficient pigs were reported in a line of Yorkshire pigs secondary to a natural mutation in the Artemis gene necessary for DNA repair during somatic mutation. SCID piglets were severely deficient in T and B cells, yet possessed granulocytes, monocytes, and natural killer cells compared with non-SCID littermates.50 Meanwhile, researchers have targeted gene mutations in RAG1, RAG2, and IL2RG in pigs to advance xenotransplanation and regenerative and transplantation medicine.51–54 Applications of Humanized Animal Models Reviews of humanized animal models, especially mouse models and their role in biomedical research, have been published.2,38,55,56 In summary, humanized animal models have been utilized in cancer biology to research the growth of tumor xenografts and patient derived xenografts (PDX), to elucidate tumor heterogeneity and surrounding stroma with PDX, to study tumor-immune system interactions, and to develop targeted cancer immunotherapy.57–60 Humanized mouse models have been utilized to study viral diseases such as Epstein Barr virus (EBV),55 HIV,55,61–63 dengue,55 herpes simplex virus,55 and hepatitis B (HBV),64 and bacterial diseases55 like Mycobacterium tuberculosis and Salmonella enterica Typhi, parasite infection56 like malaria, and sepsis.56 Humanized mouse models have also been utilized in transplantation research regarding human allografts and rejection65 and auto-immune research including Type 1 diabetes, systemic lupus erythematosus, arthritis, chronic inflammation, and allergic reactions including anaphylaxis.38 PDXs are freshly implanted human tissues or cellular suspensions (most commonly human neoplasms or fragments of a primary tumor) that have not originated from prior in vitro culture and are implanted into an immunodeficient animal. Immunodeficient mice have traditionally been utilized to study effects of tumor biology and metastasis via heterotopic or orthotopic implantation of human immortalized in vitro cancer cell lines. However, immortalized in vitro tumor cell lines do not directly recapitulate a patient’s distinct histological and molecular tumor-derived architecture, including the tumor’s stromal and microenvironment, as genomic and phenotypic characteristics may become increasingly homogeneous with repeated passaging.66 The development of humanized mouse models provides researchers with an in vivo model to evaluate the interactions and functions of the human immune system (activation or suppression) directly on a patient’s tumor and stromal microenvironment. These models can be utilized to predict a personalized and targeted response to traditional chemotherapy or novel immune-modulatory therapy. Lai et al.58 describe the methodology of patient-derived xenograft models and their role in preclinical cancer biology and cancer research. Animal Care and Humanized Animal Models Due to the complexity involved with the creation of humanized animal models, veterinary, husbandry, and Institutional Animal Care and Use Committee (IACUC), oversight is of utmost importance to achieve scientific integrity and optimize animal welfare. Humanized animal models, especially those utilizing mice, generally require preconditioning with ionizing radiation followed by engraftment of the host with human hematopoietic stem cells or tissues. As a result, pain, distress, and morbidity should be minimized as adverse effects secondary to ionizing radiation, xenogenic GVHD, or opportunistic infection may develop. Duran-Struuck and Dysko provide a thorough overview of bone marrow transplantation protocols in mice outlining methods to optimize veterinary care, husbandry, and research practices, including suggested IACUC guidance when reviewing bone marrow transplantation protocols.67 These concepts may also be applied to humanized mouse models. In summary, animal housing and husbandry care for these animals (humanized mouse models) should be based upon maintaining a strict specific pathogen-free barrier facility utilizing HEPA filtered ventilation (ventilated cages or flexible film isolator) with restricted access and appropriate PPE requirements. Housing rooms should be maintained at positive pressure to the corridor unless the animals are inoculated or implanted with hazardous biological materials. Animals should be provided autoclaved or irradiated food with acidified (pH 2.5–3), reverse osmosis, or autoclaved water. Fluid and hydration maintenance requirements (especially postirradiation), dietary hydration gel supplements, and/or subcutaneous isotonic fluids may be administered in addition to the primary water source.67 From an animal welfare perspective, the IACUC, in partnership with the veterinary team, should review each protocol involving humanized models to ensure pain and distress are minimized and humane endpoints are established. For surgical procedures or procedures that generate more than momentary or slight pain, analgesics should be administered (unless withheld as indicated by scientific justification), and environmental enrichment should be provided. Toth and Wallace described and outlined guidelines for defining experimental endpoints for animal research, including humane endpoints for cancer research, which can be applied to humanized mouse models.68,69 The goal of humane endpoints should be to promote the scientific validity of data while minimizing harm to the animal. When predicting a moribund or humane endpoint, considerations should include monitoring for hypothermia, ability to ambulate, days of consecutive weight loss, and research-specific biomarkers as predictors of imminent death.68 Similar to bone marrow transplantation, wasting disease, and GVHD as manifested by weight loss, skin alterations and hepatic dysfunction may develop in humanized mouse models. Thus, weight loss of 15% to 20% from baseline without a rebound in weight or a decreased body condition score should be monitored and considered as parameters for humane endpoints.67,70 Monitoring frequency for signs of humane endpoints should correlate with the expected severity of the experimental procedure and clinical condition of the animal. Occupational Health and Safety Regulatory Framework Animal care and use program leadership should carefully consider proposed in vivo activities, including the ethical, philosophical, and legal aspects associated with research incorporating human-derived substances (HDS). Similar to the scientific breakthroughs that are occurring rapidly in stem cell-based research, the guidelines and regulations surrounding this work are also quickly changing. While the institution needs to meet federal, state, and local rules and regulations generally applicable to animal research, certain guidelines, especially in the production and maintenance of humanized animals as they relate to occupational health and safety, must be considered and implemented accordingly. A discussion of the institutional risk assessment process and factors to consider when implementing policies that exceed regulatory standards is included in a following section. The 2 general laws that govern animal research in the United States are the Animal Welfare Act (AWA, 9 CFR)71 and the Health Research Extension Act of 1985 (Public Law 99-158).72 Although neither specifically address personnel safety when working with animals, it is important to abide by these laws as they apply to the institutional animal care and use program. AWA is the only federal law in the United States that requires that minimum standards of care and treatment be provided for certain animals bred for commercial sale, used in research, transported commercially, or exhibited to the public. For AWA, the term animal excludes rats of the genus Rattus and mice of the genus Mus bred for use in research.71 Since most humanized animals are mice specifically bred for research, they do not fall under the AWA jurisdiction. Meanwhile, the Health Research Extension Act of 1985 provides the legislative mandate for the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (PHS Policy), which is implemented by the Office of Laboratory Animal Welfare.73 In contrast with the AWA, the PHS policy applies to any live, vertebrate animal, including mice and rats, used or intended for use in research, research training, experimentation, biological testing, or for related purposes.73 The PHS requires that institutions that receive federal funds for animal research provide an occupational health program for employees with substantial animal contact.74 There are 2 regulatory documents pertinent to OHSPs in laboratory animal research. One document is the Guide for the Care and Use of Laboratory Animals75 by the National Research Council. The Guide is an internationally accepted primary reference on animal care and use, and its implementation is required in the United States by the PHS policy. AAALAC International, a private, nonprofit organization that promotes the humane treatment of animals in science through voluntary accreditation and assessment programs, uses the Guide as one of the 3 primary standards for evaluating animal care and use programs.76 The Guide indicates that each institution must establish and maintain an OHSP as an essential part of the overall animal care and use program, encouraging institutions to tailor needs to its specific program.75 OHSP deficiencies have consistently ranked in the top 3 AAALAC mandatory findings for correction.77 The second document that contributes to the OHSP regulatory framework related to the animal care and use program is the Occupational Health and Safety in the Care and Use of Research Animals, published in 1997 by the Committee on Occupational Safety and Health in Research Animal Facilities, Institute of Laboratory Animal Resources.74 This remains the authoritative guidance on the occupational health and safety of personnel in the animal care and use program. The Guide indicates that the OHSP must be consistent with federal, state, and local regulations.75 The federal law Occupational Safety and Health (OSH) Act of 1970 (29 CFR Chapter 15) was promulgated to protect employees from hazards in the workplace (CFR 1970).78 It applies to most private sector employers and their workers and some public sector employers and their workers in the United States. Compliance with regulations and standards under this law can be enforced either directly through the OSH Administration (OSHA) or through an OSHA-approved state plan.78 State laws on OSH and those pertaining to the use of human cells and tissues must also be followed. For example, human stem cell research in California must abide by state guidelines from the California Department of Public Health Human Stem Cell Research Program. Projects funded through the California Institute for Regenerative Medicine must adhere to their regulations. For more information on allowable human stem cell research within individual states, please refer to the National Conference of State Legislatures summary of State Stem Cell Research regulations (http://www.ncsl.org/research/health/embryonic-and-fetal-research-laws.aspx). There are 3 regulatory documents that have direct impact on the use of humanized animals in research: the OSHA Bloodborne Pathogen Standard (BPS) 29 CFR § 1910.1030,79 the Biosafety in Microbiological and Biomedical Laboratories (BMBL, 5th ed.),80 and the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines).81 The BPS is intended to protect workers in diverse settings from occupational exposure to blood and other potentially infectious materials (OPIM). BPS defines blood to include “human blood, human blood components, and products made from human blood”,79 including plasma derivatives,82 while OPIM includes human body fluids like semen, vaginal secretions, cerebrospinal fluid, saliva in dental procedures, any bodily fluid that is visibly contaminated with blood, all body fluids in situations where it is difficult or impossible to differentiate between body fluids, and any unfixed tissue or organ (other than intact skin) from a human (living or dead).79Bloodborne pathogens means pathogenic microorganisms that are present in human blood and can cause disease in humans and include, but are not limited to, HBV, hepatitis C virus, and HIV.79 The BPS requires employers to provide and ensure employees use appropriate PPE such as, but not limited to, gloves, gowns, laboratory coats, face shields or masks, and eye protection when handling human blood or OPIMs.79 OSHA exempts certain human cell lines from the BPS, like those that are procured from commercial vendors or other sources with documented testing to be free of human bloodborne pathogens and which have been protected by the employer from environmental contamination.83 Screening of the cell lines or “strains” will be for viruses characterized as bloodborne pathogens by the BPS, if the cells are capable of propagating such viruses. A human cell line is defined as in vitro or animal passaged (eg, nude mouse) cultures or human cells that fulfill traditional requirements of a cell line designation, while human cell strains are defined as cells propagated in vitro from primary explants of human tissue or body fluids that have finite lifetime (nontransformed) in tissue culture for 20 to 70 passages.83 The BMBL, issued by the Department of Health and Human Services, is considered to be the minimum standard of practice for all US laboratories that handle infectious microorganisms and hazardous biological materials. It provides information on good work practices, proper PPE, safety equipment, and laboratory facility design for each biosafety level (BSL). BMBL’s Section V (Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities)80 presents a summary table with recommended practices, PPE, and primary and secondary barrier characteristics for containment housing of animals administered biohazards. Appendix H describes potential laboratory hazards and recommended practices when working with human, nonhuman primate, and other mammalian cells and tissues.80 It indicates that all laboratory staff working with human cells and tissues be enrolled in an occupational medicine program specific for bloodborne pathogens, and all staff should work under the policies and guidelines established by the institution’s Exposure Control Plan.80 Besides HIV and the hepatitis viruses specifically mentioned as bloodborne pathogens in the BPS, the BMBL lists the following pathogens to be potentially harbored in human cells and tissues: human T-lymphotropic virus, EBV, human papilloma virus (HPV), human cytomegalovirus, and Mycobacterium tuberculosis (lung tissue).80 The NIH Guidelines describes the practices for constructing and handling recombinant and synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and cells, organisms, and viruses containing such molecules.81 Thus, the generation and use of transgenic animals in research is subject to the NIH Guidelines at any entity in receipt of NIH funds for research involving recombinant or synthetic nucleic acid molecules. Transgenic animal creation falls under various sections of the NIH Guidelines depending on the animal and the BSL required, but in most cases requires registration with, and approval from, the institutional biosafety committee (IBC). While the IBC’s primary duty is to review research using recombinant and synthetic nucleic acid molecules, most institutions expand the IBC’s authority to cover infectious disease research. Thus, the IBC can determine containment housing for such animals. The IBC should be contacted for additional information about the institutional approval process. Occupational Health and Safety Program General Components As mentioned above, OHSP is a requirement for any animal care and use program and for the use of human biologics. The Guide describes a 3-fold management approach for a robust OHSP that is a shared responsibility of several groups in the institution.75 First, engineering controls entail appropriate safety equipment provision and facility design and operation. Second, administrative controls need to be implemented to clearly describe processes and standard operating procedures. Finally, when exposure to hazards cannot be engineered completely out of normal operations and when safe work practices and other forms of administrative controls cannot provide sufficient additional protection, the use of personal PPE provides a supplementary means of control and serves as the last line of defense for risk exposure. The reader is directed to the Guide75 and chapter 6 of the Occupational Health and Safety in the Care and Use of Research Animals74 for more detailed information on the principal components of an OHSP. The review article by Dyson et al.84 also provides additional information on the institutional oversight of occupational health and safety for research programs involving biohazards. Programmatic components like exposure control, disaster plans, health surveillance, training and education, and information management are discussed in the Dyson paper. Lastly, the review article of Villano et al.85 on PPE gives readers a knowledge base for evaluating the adequacy and effectiveness of institutional PPE requirements by providing a comprehensive review of risk assessment, common PPE used in laboratory animal research, and PPE standards and regulations. Training and education is an integral component of OHSP. One training specifically needed for the use of HDS should be focused on bloodborne pathogens. This should be given for all personnel who may reasonably anticipate contact with human blood, blood products, tissues, fluids, or OPIM including human cell lines. The BPS requires employers to ensure that workers receive regular training that covers all elements of the standard, including but not limited to information on bloodborne pathogens and diseases, methods used to control occupational exposure like safe handling of sharps and wastes, HBV vaccinations, and medical evaluation, including postexposure follow-up procedures like injury reporting.79 Such training must be provided on initial assignment, at least annually thereafter, and when new or modified tasks or procedures affect a worker’s risk of occupational exposure.79 Other topics for training that are strongly recommended include donning and doffing procedures of PPE, spill management, and working safely with relevant animal species. Training on these topics can particularly be effective in a practical setting and in conjunction with or as prerequisites for a facility orientation. It is important to emphasize that OHSP is a shared responsibility of several groups in the institution, including the IACUC, the IBC, veterinary and husbandry staff, researchers, the environmental health and safety (EHS) unit, and OHS healthcare workers such as physicians and nurses (Figure 1). A close collaboration among these groups is necessary for creation and implementation of policies, guidelines, and standard operating procedures that cover risk assessment, provision of engineering controls and PPE, practices, medical treatment and intervention, and education and training. Figure 1 Open in new tabDownload slide According to the Guide for the Care and Use of Laboratory Animals (NCR 2011), coordination among 5 groups in the institution is needed for an effective OHSP. 1RO = responsible official. This is the individual at the entity who is accountable for entity compliance with the select agent regulations. Adopted from Villano and Ogden96 with permission. Figure 1 Open in new tabDownload slide According to the Guide for the Care and Use of Laboratory Animals (NCR 2011), coordination among 5 groups in the institution is needed for an effective OHSP. 1RO = responsible official. This is the individual at the entity who is accountable for entity compliance with the select agent regulations. Adopted from Villano and Ogden96 with permission. This shared responsibility can present contemporary challenges such as the formation of departmental silos or independent strategies that can prevent these entities from working efficiently. Successful programs should employ multiple measures by which the IBC, IACUC, AV, and EHS and OHSP personnel can foster effective communication, especially with researchers. An animal containment expert, typically the AV or his/her designee, must be a member of the IBC, per the NIH Guidelines.81 This allows for the animal care program and the IBC to be abreast of current and new challenges that face each group together and independently. Some IACUCs also incorporate an EHS personnel as a committee member or a nonvoting consultant so that she or he can provide valuable occupational health insight to committee members in real time. Finally, IACUCs can integrate the abovementioned groups by having representatives of the IBC, animal care and use program, and EHS/OHSP provide IACUC member training exercises. Management of research applications, that is, IACUC protocol and corresponding IBC application, by the IACUC and IBC, respectively, with input and involvement of EHS personnel may also be discussed and improved. It is important to ensure congruency in the description of animal experiments as described in an IBC application and an IACUC protocol. Although one does not need to be contingent upon the other, having the IBC application approved prior to IACUC protocol approval ensures that risk assessment has been performed and risk mitigation is in place prior to commencement of animal experiments. Risk Assessment, Guidelines, and Recommendations Risk assessment is the first step to align the institutional OHSP to personnel safety. This evaluates the workplace to identify hazards and the risks associated with those hazards and determines the appropriate measures that should be in place to effectively eliminate or control the hazard.85 It carefully evaluates the facility and its equipment and bridges the gap between engineering and administrative controls.85 Additionally, personnel should be medically evaluated based on several factors, including special conditions like pregnancy and immune status. The nature of activities, especially the potential for aerosolization, is a significant consideration for risk assessment. For example, procedures that entail direct handling of primary human cells or tissues, such as their implantation into animals, can pose higher risk than routine husbandry procedures of animals administered such substances. The final outcome of risk assessment is the provision of engineering standards and PPE and establishment of safety practices based on the appropriate containment level as dictated by hazard identification. This outcome is to be made collectively by a team that includes safety professionals, occupational health professionals, and veterinary and husbandry personnel and should include input from research personnel.85 A strong hazard analysis program is dependent on not just identifying and mitigating the risks, but communicating and training the staff of the hazards identified and the controls implemented.85 Institutional risk assessment processes pertaining to humanized animals should include the regulatory guidelines produced by the Guide, OSHA BPS, BMBL, and the NIH Guidelines. While the minimum regulatory requirements must be met, some institutions implement policies that extend beyond these requirements. An example of this relates to the exemption of some human cell lines from the BPS. A concern with perpetual exemption of these cell lines occurs when cells are obtained from a source other than the original vendor or proprietor. Over time, cell lines can become contaminated either by infectious agents or with other cell lines. Institutions that follow the OSHA exemptions for established cell lines should consider best practices to verify or test incoming cell lines or perform periodic validation on current cell lines. In contrast, some institutions include the use of all primary and established cell lines within the scope of their BPS oversight, and some institutions also include all nonhuman primate cells, blood, blood products, tissues, etc., as they pose both a zoonotic risk relating to Macacine herpersvirus 1 (herpes B virus) but also are capable of supporting replication of some common human bloodborne pathogens. The risk assessment for this may incorporate the idea that the list of commonly tested bloodborne pathogens is not comprehensive, there are not reliable tests for all known agents, and not all potential agents have been identified. The institutional inclusion of all HDS within BPS oversight leads to a second example of regulatory extension: the determination of BSL for use of human cells and tissues. Some institutions require that all human cells, tissues, blood, and blood products not known or suspected to contain biohazardous agents be handled under the approach termed universal precautions. Universal precautions employs infection control practices by handling these materials as if they were potential positive for bloodborne pathogens. Alternatively, some institutions require that all human materials be handled at BSL2, with or without the possibility of downgrading to BSL1 upon official review of necessary documentation. There is a lot of overlap between these 2 types of policies (eg, BBP training, PPE usage, aerosol precautions), but incorporation and oversight of users pose different challenges. A risk-based assessment like universal precautions and the elevation of some cells, such as liver carcinoma cells that have a high prevalence of hepatitis, to a higher BSL is a flexible and customizable approach but requires more effort to ensure compliance across the board if there is no formal IBC or institutional oversight. A blanket approach, such as categorical BSL2 assignment, allows for improved oversight by an IBC or other group but requires more effort for full review and may result in push-back from researchers requiring justification. The risk associated with humanizing animals and the use of such animals should be carefully evaluated by the institution. The BMBL specifically states that each institution should conduct this assessment based on the origin of the cells or tissues (species and tissue type) as well as the source (recently isolated or well characterized).80 Understanding the human materials these animals have and how these affect the animal’s physiology will help determine the risks involved. However, experimental manipulations, especially inoculating these humanized animals with infectious agents, may increase the risk and will thus likely be the final determinant of containment level for animal handling and housing. Therefore, the institution needs to perform risk assessment on 2 stages when working with HDS: Handling (in vitro) HDS can either be primary (direct patient-derived) or secondary (commercial vendors). All laboratory work with primary human tissues or body fluids is covered by the BPS,83 and these may be considered high risk especially if obtained from patients infected with bloodborne pathogens (eg, liver tumor samples from patients with HBV). As such, engineering and work practice controls should be used to eliminate or minimize employee exposure, and where occupational exposure remains after institution of these controls, PPE shall also be used.79 As mentioned above, however, HDS that are characterized by documented, reasonable laboratory testing to be free of bloodborne pathogens may be exempted from the BPS. This documentation is also necessary for human cervical carcinoma cells or other transformed human cell lines like HeLa cells as they are sometimes adulterated with laboratory pathogens accidentally introduced by cultivation with other cell cultures or physically contaminated by other cell cultures handled in the same laboratory.83 Recent reports from 2 diagnostic laboratories indicate that EBV86,87 and HPV1687 were the most common among a wide variety of pathogens in human samples submitted. The documentation that such cell lines are not OPIM should be a matter of written record and on file with the employer for OSHA review,83 though institutional risk assessment and best practice may still result in some of these cell lines remaining included in the BPS. Regardless of inclusion under the BPS umbrella, best practice should dictate that all HDS be handled with appropriate precautions due to the unknown potential for bloodborne pathogens not tested for. It is helpful to review available information for any HDS obtained from commercial vendors, especially the appropriate BSL that may differ from material to material. For example, BSL1 is typically sufficient for handling cell lines derived from normal tissue of healthy patients, while cells that are transformed by natural or laboratory infection with an immortalizating agent such as EBV would require BSL2 containment. The American Type Culture Collection (ATCC), the premier global biological materials resource and standards organization, has all human cell lines accessioned in its general collection tested for HIV, HBV, hepatitis C virus (until August 2012), HPV, EBV, and human cytomegalovirus.88 The decision to remove hepatitis C from the ATCC virus panel test was based on the reason that there are no known culture cell lines that support the replication of this virus.88 The institution can choose to use a virus screen panel similar to what ATCC uses to permit BSL-1 conditions. It is of note that the BMBL indicates that human and other primate cells should be handled using BSL-2 practices and containment, and that all work should be performed in a biosafety cabinet and all material decontaminated by autoclaving or disinfection before discarding.80 Also of note, the correct BSL for human pathogens is determined by the CDC and/or NIH if human pathogens or rDNA is involved, and the commercial designations are not always in agreement with the CDC or with each other, particularly if commercial vendors are located in different countries. For example, while the ATCC lists Raji cells as BSL-289 due to the presence of EBV (a risk group [RG] 2 agent per CDC standard), the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures lists Raji cells as BSL-1.90 The final judgement for making the determination that human or other animal cell lines in culture are free of bloodborne pathogens must be made by a biosafety professional or other qualified scientist with the background and experience to review such potential contamination and risk, in accordance with the requirements of the BPS.83 Administration to animals and animal housing (in vivo) The containment level for handling an HDS also determines practices for its administration to animals but does not necessarily dictate animal housing requirements. There are several risk assessment factors to consider in determining the appropriate containment level for administration to animals and animal housing. These include the animal’s pathogen status, species-specific behavior, route of administration, personnel training, and the use of other hazards, all of which will determine necessity for additional practices to ensure personnel safety. For example, a subcutaneous injection of an HDS to a manually restrained, specific-pathogen free mouse may be performed by staff proficient in the procedure, but administration of a human pathogen to a humanized mouse may necessitate the use of restraint devices or anesthesia. It is important to secure the animal during the HDS administration, as any animal movement may cause a spill and aerosolization or equipment movement and a sharps injury, possibly exposing personnel. Certain surgical procedures that involve implantation of human cells or tissues into an animal’s living system and that require extensive manipulation like those involving sharps or the bones (orthopedic surgery) may pose a high risk for aerosolization or punctures/lacerations. PPE requirements and the use of engineering standards like biosafety cabinets and safety sharps should be evaluated based on personnel exposure risk. Animals administered unmodified and established human cell lines, especially those documented to be free of bloodborne pathogens, may be housed under animal biosafety level (ABSL)-1.91 As stated above, the institution, through the IBC, may define a human pathogen screening panel like the ATCC’s. Meanwhile, ABSL-2 housing is appropriate for humanized animals like the hu-CD34 mice, for animals that carry primary human tissues or body fluids,36,91 or those administered human materials known to be infected with human pathogens, and if the planned host animal is transgenic for receptors or other genetic loci that could enable infection, replication, or shedding of human pathogens inoculated onto the animal host. Some institutions include in BSL-2/ABSL-2 the category of human material suspected to be infected with human pathogens, with examples including hepatocellular or cervical carcinoma cells based on the high prevalence of hepatitis virus or HPV in these samples. The use of human cells transduced with viral vectors can also impact containment level for administration and animal housing requirements, and this needs to be reviewed by the IBC with the help of an animal expert81 and using the NIH Guidelines. IBC considerations should, at a minimum, include review of the tropism of the virus for human or animal cells (eg, adenoviral vector administered to an animal with human cells), pseudotyping of the viral vector to expand cell tropism or host range (eg, use of the VSV-g envelope on a nonhuman viral vector), and the transgene of interest (eg, oncogene or toxin). Collins et al.92 reviewed the most commonly used viral vectors in animal research. NIH classifies most viral vectors as either RG1 (not associated with disease in healthy human adults) or RG2 (associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available); BSL containment requirements are stipulated for these groups as BSL-1 (BL1) or BSL-2 (BL2), respectively.81 Viral vectors containing less than two-thirds of a eukaryotic viral genome may be handled under BL1 conditions.92 Most viral vectors used in animal research are either RG2 agents or do not meet this size requirement and therefore require BL2 containment and procedures during preparation, manipulation, and injection.92 These precautions include restricted access, an appropriate laboratory set-up and signage, staff training, sharps safety, decontamination of waste prior to disposal, and PPE to prevent skin and mucous membrane exposure.93 All these safety practices are generally recommended for HDS administration to animals. Following these practices for humanized animals may often have additional benefits in that maintaining occupational health-related housing and precautions can aid general animal health upkeep, particularly for viral vectors that are zoonotic or are based on animal pathogens. For animal experiments involving most viral vectors used in research, the NIH Guidelines recommend ABSL-2 containment practices.81 Both lentiviral and adenoviral vectors can be found on tail swabs for as long as 72 hours after injection, although this positivity was localized and no vector was recovered from the bedding.94 Although viral vectors are rapidly cleared (ie, within 24 hours) from the blood, the injection site should then be wiped with a disinfectant to further minimize the risk of environmental contamination.94 The route of delivery of the transduced cells can also impact housing requirements. For example, intracranial delivery methods are designed as closed injection systems that use small volumes such that the potential of superficial contamination is minimal when properly performed.92 Further clarification from Department of Health and Human Services indicates that ABSL2 containment housing for 1 to 7 days may be permissible for lentiviral vectors.95 This system is also applicable for standard replication-deficient adenoviral and third-generation herpesviral vectors.92 Experimental manipulations of HDS-administered animals may ultimately dictate containment level housing. This is especially true when humanized animals are used as models of infectious disease, in which case BMBL animal housing requirements need to be followed and, depending on the IBC charge, an IBC application is needed. Animal tissues known to be contaminated by deliberate infection with HIV or HBV are also subject to the BPS.83 Conclusions The use of humanized animals provides unique challenges for the institutional animal care and use program and OHSP. Their use comes with the potential for personnel exposure to hazards within the human material, such as bloodborne pathogens, or other hazards like infectious agents and viral vectors as a result of further experimental manipulation of these animal models. Cornerstones for mitigating risks involve thorough risk assessment, appropriate practices, PPE, engineering standards appropriate for the containment level, and personnel education and training, especially relating to bloodborne pathogens. The institution must have a mechanism in place to ensure safety of all personnel working with humanized animals, one that addresses both animal health and welfare issues and safety requirements for working with animals as well as HDS. 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TI - Safety Considerations When Working with Humanized Animals
JF - ILAR Journal
DO - 10.1093/ilar/ily012
DA - 2018-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/safety-considerations-when-working-with-humanized-animals-nkDyxaI1yz
SP - 150
VL - 59
IS - 2
DP - DeepDyve
ER -